Compositions and therapeutic methods using morphogenic proteins and stimulatory factors

ABSTRACT

The present invention provides pharmaceutical compositions comprising a morphogenic protein stimulatory factor (MPSF) for improving the tissue inductive activity of morphogenic proteins, particularly those belonging to the BMP protein family. Methods for improving the tissue inductive activity of a morphogenic protein in a mammal using those compositions are provided. This invention also provides implantable morphogenic devices comprising a morphogenic protein and a MPSF disposed within a carrier, that are capable of inducing tissue formation in allogeneic and xenogeneic implants. Methods for inducing local tissue formation from a progenitor cell in a mammal using those devices are also provided. A method for accelerating allograft repair in a mammal using morphogenic devices is provided. This invention also provides a prosthetic device comprising a prosthesis coated with a morphogenic protein and a MPSF, and a method for promoting in vivo integration of an implantable prosthetic device to enhance the bond strength between the prosthesis and the existing target tissue at the joining site. Methods of treating tissue degenerative conditions in a mammal using the pharmaceutical compositions are also provided.

BACKGROUND OF THE INVENTION

Osteogenic proteins were defined originally as an activity present inmammalian bone extracts, presumably active during growth and naturalbone healing, capable of inducing a developmental cascade leading tocartilage and endochondral bone accumulation when implanted in vivo.This developmental cascade includes mesenchymal cell recruitment andproliferation, progenitor cell differentiation, cartilage calcification,vascular invasion, bone formation, remodeling and marrow differentiation(Reddi, Collagen Rel. Res., 1, pp. 209-26 (1981)).

The factors in bone matrix that induce endochondral bone differentiationcan be dissociatively extracted and reconstituted with inactivecollagenous matrix to restore full bone inductive activity (Reddi, Proc.Natl. Acad. Sci. USA, 78, pp. 7599-7603 (1981)). This provides anexperimental method for assaying protein extracts for their ability toinduce endochondral bone formation in vivo. Using this reconstitutionassay, a variety of related osteogenic proteins have been isolated fromseveral mammalian species that are capable of inducing bone andcartilage formation in cross-species implants (Sampath and Reddi, Proc.Natl. Acad. Sci. USA, 80, pp. 6591-95 (1983)). The active factor orfactors that promote this activity have been referred to in theliterature most commonly as bone morphogenetic proteins (BMPs) andosteogenic proteins (OPs).

Osteogenic and bone morphogenetic proteins represent a family ofstructurally and functionally related morphogenic proteins belonging tothe Transforming Growth Factor-Beta (TGF-β) superfamily (see below). TheTGF-β superfamily, in turn, represents a large number of evolutionarilyconserved proteins with diverse activities involved in growth,differentiation and tissue morphogenesis and repair. BMPs and osteogenicproteins, as members of the TGF-β superfamily, are expressed assecretory polypeptide precursors which share a highly conservedbioactive cysteine domain located near their C-termini. Another featureof many of the BMP family proteins is their propensity to form homo- andheterodimers.

Many morphogenic proteins belonging to the BMP family have now beendescribed. Some have been isolated using purification techniques coupledwith bioassays such as the one described above. Others have beenidentified and cloned by virtue of DNA sequence homologies withinconserved regions that are common to the BMP family. These homologs arereferred to as consecutively-numbered BMPs whether or not they havedemonstrable osteogenic activity. Using an alternative approach,synthetic OPs having osteogenic activity have been designed using aminoacid consensus sequences derived from sequence comparisons betweennaturally-derived OPs and BMPs (see below; Oppermann et al., U.S. Pat.No. 5,324,819).

While several of the earliest members of the BMP family were osteogenicproteins identified by virtue of their ability to induce new cartilageand bone, the search for BMP-related genes and gene products in avariety of species has revealed new morphogenic proteins, some of whichhave different or additional tissue-inductive capabilities. For example,BMP-12 and BMP-13 (identified by DNA sequence homology) reportedlyinduce tendon/ligament-like tissue formation in vivo (WO 95/16035).Several BMPs can induce neuronal cell proliferation and promote axonregeneration (WO 95/05846). And some BMPs that were originally isolatedon the basis of their osteogenic activity also have neural inductiveproperties (Liem et al., Cell, 82, pp. 969-79 (1995)). It thus appearsthat osteogenic proteins and other BMPs may have a variety of potentialtissue inductive capabilities whose final expression may depend on acomplex set of developmental and environmental cues. These osteogenic,BMP and BMP-related proteins are referred to herein collectively asmorphogenic proteins.

The activities described above, and other as yet undiscovered tissueinductive properties of the morphogenic proteins belonging to the BMPfamily are expected to be useful for promoting tissue regeneration inpatients with traumas caused, for example, by injuries or degenerativedisorders. Implantable osteogenic devices comprising mammalianosteogenic protein for promoting bone healing and regeneration have beendescribed (see, e.g., Oppermann et al., U.S. Pat. No. 5,354,557). Someosteogenic devices comprise osteogenic protein dispersed in porous,biocompatible matrices. These naturally-derived or synthetic matricestypically allow osteogenic protein to diffuse out of the matrix into theimplantation site and permit influx and efflux of cells. Osteogenicprotein induces the progenitor cells to differentiate and proliferate.Progenitor cells may migrate into the matrix and differentiated cellscan move out of the porous matrix into the implant site. Osteogeniccells may also utilize the matrix as a physical scaffold forosteoconduction. Similar devices have been described for delivering BMPsfor tendon/ligament-like and neural tissue regeneration (see below).Osteogenic protein-coated prosthetic devices which enhance the bondstrength between the prosthesis and existing bone have also beendescribed (Rueger et al., U.S. Pat. No. 5,344,654, incorporated hereinby reference).

The availability of large amounts of purified and highly activemorphogenic proteins would revolutionize orthopedic medicine, certaintypes of plastic surgery, dental and various periodontal andcraniofacial reconstructive procedures, and procedures generallyinvolving bone, cartilage, tendon, ligament and neural regeneration.Many of the mammalian OP- and BMP-encoding genes are now cloned and maybe recombinantly expressed as active homo- and heterodimeric proteins ina variety of host systems, including bacteria. The ability torecombinantly produce active forms of morphogenic proteins such as OPsand BMPs, including variants and mutants with increased bioactivities(see below), make potential therapeutic treatments using morphogenicproteins feasible.

Given the large number of potential therapeutic uses for morphogenicproteins in treating a variety of different tissues and tissue-types,there is a need for highly active forms of morphogenic proteins. Itwould thus be desirable to increase the tissue inductive properties ofmorphogenic proteins. With increased tissue inductive activity,treatment with a morphogenic protein, even on large scales, could inducetissue formation more rapidly, or tissue induction could be achievedusing reduced morphogenic protein concentrations.

SUMMARY OF THE INVENTION

The present invention solves these problems by providing pharmaceuticalcompositions comprising a morphogenic protein stimulatory factor (MPSF)for improving the tissue inductive activity of a morphogenic protein,particularly one belonging to the BMP protein family such as osteogenicprotein. Methods for improving the tissue inductive activity of amorphogenic protein in a mammal using those compositions are provided.This invention also provides implantable morphogenic devices, comprisinga morphogenic protein and a MPSF disposed within a carrier, that arecapable of inducing tissue formation in allogeneic and xenogeneicimplants. Methods for inducing local tissue formation from a progenitorcell in a mammal using those compositions and devices are also provided.A method for accelerating allograft repair in a mammal using thosemorphogenic devices is provided. This invention also provides aprosthetic device comprising a prosthesis coated with a morphogenicprotein and a MPSF, and a method for promoting in vivo integration of animplantable prosthetic device to enhance the bond strength between theprosthesis and the existing target tissue at the joining site. Methodsfor treating tissue degenerative conditions in a mammal using thepharmaceutical compositions are also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. IGF-I is a MPSF that stimulates OP-1 osteogenic induction.Alkaline phosphatase (AP) activity (nmol/μg protein) in FRC cells isplotted as a function of increasing IGF-I concentrations (ng/ml) in thepresence or absence of OP-1 (500 ng/ml).

FIG. 2. Anti-IGF-I monoclonal antibodies inhibit IGF-I stimulation ofOP-1 osteogenic induction. FRC cells were incubated with a monoclonalantibody (Upstate Biotech) against IGF-I for 48 hours in the presence orabsence of OP-1 (500 ng/ml). The level of alkaline phosphatase (nmol/μgprotein) in each culture was measured.

FIG. 3. IGF-I and OP-1 dose response curves for bone inductive activity.Relative alkaline phosphatase (AP) activity (%) in FRC cells is plottedas a function of increasing IGF-I concentrations (purchased from BRL;0-100 ng/ml) in the absence or presence of increasing concentrations ofOP-1 (0-500 ng/ml).

FIG. 4. Timing of OP-1 and IGF-I addition. Alkaline phosphatase (AP)activity (nmol/μg protein) in FRC cells is indicated. FRC cells weregrown in serum free media containing 500 ng/ml OP-1, and IGF-I (25ng/ml) was added to the culture at different times (hours) subsequently.Control cultures were grown in serum free media containing solventvehicles.

FIG. 5. Estradiol is a MPSF in concert with OP-1. Alkaline phosphatase(AP) activity (nmol/μg protein) in FRC cells is indicated. FRC cellswere grown in serum free media containing OP-1 alone (200 ng/ml), orcontaining increasing concentrations of estradiol (0.05, 0.5 and 5.0 nM)in the presence or absence of 200 ng/ml OP-1. Control cultures (CON)were grown in serum free media containing solvent vehicles.

FIG. 6. Growth hormone is a MPSF in concert with OP-1. Alkalinephosphatase (AP) activity (nmol/μg protein) in FRC cells is indicated.FRC cells were incubated in serum free media containing OP-1 alone (200ng/ml; "0"), or containing increasing concentrations of hGH (10-100ng/ml) in the presence of 200 ng/ml OP-1. Control cultures (CON) weregrown in serum free media containing solvent vehicles (not shown).

FIG. 7. Hydrocortisone is a MPSF in concert with OP-1. Alkalinephosphatase (AP) activity (nmol/μg protein) in FRC cells is indicated.FRC cells were incubated in serum free media containing OP-1 alone (200ng/ml), or containing increasing concentrations of hydrocortisone (0.05,0.5 and 5.0 nM) in the presence or absence of 200 ng/ml OP-1. Controlcultures (CON) were grown in serum free media containing solventvehicles.

FIG. 8. Insulin is a MPSF in concert with OP-1. Alkaline phosphatase(AP) activity (nmol/μg protein) in FRC cells is indicated. FRC cellswere incubated in serum free media containing OP-1 alone (200 ng/ml), orcontaining increasing concentrations of insulin (0.05, 0.5 and 5.0 nM)in the presence or absence of 200 ng/ml OP-1. Control cultures (CON)were grown in serum free media containing solvent vehicles.

FIG. 9. Parathyroid hormone is a MPSF in concert with OP-1. Alkalinephosphatase (AP) activity (nmol/μg protein) in FRC cells is indicated.FRC cells were incubated with OP-1 alone (200 ng/ml) and with increasingconcentrations of parathyroid hormone (PTH; 25, 100 and 200 ng/ml) inthe presence or absence of 200 ng/ml OP-1. Control cultures (CON) weregrown in serum free media containing solvent vehicles.

FIG. 10. Progesterone is a MPSF in concert with OP-1. Alkalinephosphatase (AP) activity (nmol/μg protein) in FRC cells is indicated.FRC cells were incubated with OP-1 alone (200 ng/ml) and with increasingconcentrations of progesterone (0.05, 0.5 and 5.0 nM) in the presence orabsence of 200 ng/ml OP-1. Control cultures (CON) were grown in serumfree media containing solvent vehicles.

FIG. 11. IGF-II does not stimulate OP-1-induced osteogenic induction.Alkaline phosphatase (AP) activity (nmol/μg protein) in FRC cells isindicated. FRC cells were incubated with OP-1 alone (500 ng/ml) and withincreasing concentrations of IGF-II (10-300 ng/ml) in the presence orabsence (shown only for 100 and 200 ng/ml IGF-II) of 500 ng/ml OP-1.Control cultures (CON) were grown in serum free media containing solventvehicles.

FIG. 12. TGF-β does not stimulate OP-1-induced osteogenic induction.Alkaline phosphatase (AP) activity (nmol/μg protein) in FRC cells isindicated. FRC cells were grown in serum free media containing: OP-1alone (200 ng/ml), TGF-β alone (0.05-2 ng/ml), or containing increasingconcentrations of TGF-β (0.05-50 ng/ml) in the presence of OP-1 at 100ng/ml, 200 ng/ml or 500 ng/ml. Control cultures (CON) were grown inserum free media containing solvent vehicles.

DETAILED DESCRIPTION OF THE INVENTION

In order that the invention herein described may be fully understood,the following detailed description is set forth.

The term "biocompatible" refers to a material that does not elicitdetrimental effects associated with the body's various protectivesystems, such as cell and humoral-associated immune responses, e.g.,inflammatory responses and foreign body fibrotic responses. The termbiocompatible also implies that no specific undesirable cytotoxic orsystemic effects are caused by the material when it is implanted intothe patient.

The term "bone morphogenetic protein (BMP)" refers to a proteinbelonging to the BMP family of the TGF-β superfamily of proteins (BMPfamily) based on DNA and amino acid sequence homology. A protein belongsto the BMP family according to this invention when it has at least 50%amino acid sequence identity with at least one known BMP family memberwithin the conserved C-terminal cysteine-rich domain which characterizesthe BMP protein family. Members of the BMP family may have less than 50%DNA or amino acid sequence identity overall.

The term "morphogenic protein" refers to a protein having morphogenicactivity (see below). Preferably a morphogenic protein of this inventioncomprises at least one polypeptide belonging to the BMP protein family.Morphogenic proteins may be capable of inducing progenitor cells toproliferate and/or to initiate differentiation pathways that lead tocartilage, bone, tendon, ligament, neural or other types of tissueformation depending on local environmental cues, and thus morphogenicproteins may behave differently in different surroundings. For example,an osteogenic protein may induce bone tissue at one treatment site andneural tissue at a different treatment site.

The term "osteogenic protein (OP)" refers to a morphogenic protein thatis capable of inducing a progenitor cell to form cartilage and/or bone.The bone may be intramembranous bone or endochondral bone. Mostosteogenic proteins are members of the BMP protein family and are thusalso BMPs. However, the converse may not be true. BMPs (identified bysequence homology) must have demonstrable osteogenic activity in afunctional bioassay to be osteogenic proteins according to thisinvention.

The term "morphogenic protein stimulatory factor (MPSF)" refers to afactor that is capable of stimulating the ability of a morphogenicprotein to induce tissue formation from a progenitor cell. The MPSF mayhave a direct or indirect effect on enhancing morphogenic proteininducing activity. For example, the MPSF may increase the bioactivity ofanother MPSF. Agents that increase MPSF bioactivity include, forexample, those that increase the synthesis, half-life, reactivity withother biomolecules such as binding proteins and receptors, or thebioavailability of the MPSF.

The terms "morphogenic activity", "inducing activity" and "tissueinductive activity" alternatively refer to the ability of an agent tostimulate a target cell to undergo one or more cell divisions(proliferation) that may optionally lead to cell differentiation. Suchtarget cells are referred to generically herein as progenitor cells.Cell proliferation is typically characterized by changes in cell cycleregulation and may be detected by a number of means which includemeasuring DNA synthetic or cellular growth rates. Early stages of celldifferentiation are typically characterized by changes in geneexpression patterns relative to those of the progenitor cell, which maybe indicative of a commitment towards a particular cell fate or celltype. Later stages of cell differentiation may be characterized bychanges in gene expression patterns, cell physiology and morphology. Anyreproducible change in gene expression, cell physiology or morphologymay be used to assess the initiation and extent of cell differentiationinduced by a morphogenic protein.

The term "synergistic interaction" refers to an interaction in which thecombined effect of two agents is greater than the algebraic sum of eachof their individual effects.

Morphogenic proteins

The morphogenic proteins of this invention are capable of stimulating aprogenitor cell to undergo cell division and differentiation, and thatinductive activity may be enhanced in the presence of a MPSF. Manymammalian morphogenic proteins have been described. Some fall within aclass of products called "homeodomain proteins", named for theirhomology to the drosophila homeobox genes involved in phenotypicexpression and identity of body segments during embryogenesis. Othermorphogenic proteins are classified as peptide growth factors, whichhave effects on cell proliferation, cell differentiation, or both.

Peptide growth factors may be grouped into a number of superfamilies orfamilies based primarily on their sequence similarity (Mercola andStiles, Development, 102, pp. 461-60 (1988)). These families include:Epidermal Growth Factor (e.g., EGF, TGF-α, notch and delta),Transforming Growth Factor-Beta (e.g., TGF-β, inhibin, activin, MIS,BMP, dpp and Vg-1); Heparin Binding Growth Factor (e.g., FGF, ECDGF andint-2); Platelet Derived Growth Factor; Insulin-like Growth Factor(IGF-I, IGF-II); and Nerve Growth Factor.

The BMP Family

The morphogenic proteins of this invention whose activity may beenhanced in the presence of a MPSF preferably belong to the TGF-βprotein superfamily. Members of the TGF-β superfamily are dividedfurther into families based on their degree of structural or functionalsimilarity. The BMP family is one such family, named for itsrepresentative bone morphogenetic/osteogenic protein family members. Ofthe reported "BMPs" (BMP-1 to BMP-13), isolated primarily based onsequence homology, all but BMP-1 remain classified as members of the BMPfamily of morphogenic proteins (Ozkaynak et al., EMBO J., 9, pp. 2085-93(1990)).

The BMP family includes other structurally-related members which aremorphogenic proteins, including the drosophila decapentaplegic genecomplex (DPP) products, the Vg1 product of Xenopus laevis and its murinehomolog, Vgr-1 (see, e.g., Massague, J., "The Transforming GrowthFactor-β Family", Annu. Rev. Cell Biol., 6, pp. 597-641 (1990)).

A morphogenic protein according to this invention belongs to the BMPfamily when it comprises a polypeptide having at least 50% amino acidsequence identity with at least one known BMP family member, within theconserved C-terminal cysteine-rich domain which characterizes the BMPprotein family. This definition is in part derived from comparing aminoacid sequence identities between C-terminal domains of other BMP familymembers that have demonstrable morphogenic activity.

The Drosophila DPP and Xenopus Vg-1 gene products are 50% identical toeach other (and 35-40% identical to TGF-β). Both the Dpp and Vg-1products are morphogenic proteins that participate in early patterningevents during embryogenesis of their respective hosts. These productsappear to be most closely related to mammalian bone morphogeneticproteins BMP-2 and BMP-4, whose C-terminal domains are 75% identicalwith that of Dpp.

The C-terminal domains of BMP-3, BMP-5, BMP-6, and OP-1 (BMP-7) areabout 60% identical to that of BMP-2, and the C-terminal domains ofBMP-6 and OP-1 are 87% identical. BMP-6 is likely the human homolog ofthe murine Vgr-1 (Lyons et al., Proc. Natl. Acad. Sci. U.S.A., 86, pp.4554-59 (1989)); the two proteins are 92% identical overall at the aminoacid sequence level (U.S. Pat. No. 5,459,047, incorporated herein byreference). BMP-6 is 58% identical to the Xenopus Vg-1 product.

The DNA and amino acid sequences of these and other BMP family membersare published and may be used by those of skill in the art to determinewhether a new candidate gene product belongs to the BMP family. NewBMP-related gene products are expected by analogy to possess at leastone morphogenic activity.

Another characteristic of the BMP protein family members is theirapparent ability to dimerize. Several bone-derived osteogenic proteins(OPs) and BMPs are found as homo- and heterodimers in their activeforms. The ability of OPs and BMPs to form heterodimers may conferadditional or altered morphogenic inductive capabilities on morphogenicproteins. Heterodimers may exhibit qualitatively or quantitativelydifferent binding affinities than homodimers for OP and BMP receptormolecules. Altered binding affinities may in turn lead to differentialactivation of receptors that mediate different signalling pathways,which may ultimately lead to different biological activities oroutcomes. Altered binding affinities could also be manifested in atissue or cell type-specific manner, thereby inducing only particularprogenitor cell types to undergo proliferation and/or differentiation.

Suitable in vitro, ex vivo and in vivo bioassays known in the art,including those described herein, may be used to ascertain whether a newBMP-related gene product or a new heteromeric species has a known or anew morphogenic activity. Expression and localization studies definingwhere and when the gene and its product(s) are expressed may also beused to identify potential morphogenic activities. Nucleic acid andprotein localization procedures are well known to those of skill in theart (see, e.g., Ausubel et al., eds. Current Protocols in MolecularCloning, Greene Publishing and Wiley Interscience, New York, 1989).

Many of the identified BMPs are osteogenic and can induce bone andcartilage formation when implanted into mammals. Some BMPs identifiedbased on sequence homology to osteogenic proteins possess othermorphogenic activities and the MPSFs according to this invention may beused to enhance those other activities. For example, BMP-12 and BMP-13reportedly induce ectopic formation of tendon/ligament-like tissue whenimplanted into mammals (Celeste et al., WO 95/16035). Using thisbioassay, or any other suitable assay selected by the skilledpractitioner, one or more MPSFs that are capable of stimulating theability of the BMP to induce tendon/ligament-like tissue formation canbe identified and optimized according to the procedures describedherein.

Certain BMPs which are known to be osteogenic can also induce neuronalcell differentiation. Embryonic mouse cells treated with BMP-2 or OP-1(BMP-7) differentiate into astrocyte-like (glial) cells, and peripheralnerve regeneration using BMP-2 has been recently reported (Wang et al.,WO 95/05846). In addition, BMP-4, BMP-5 and OP-1 (BMP-7) are expressedin epidermal ectoderm flanking the neural plate. Ectopic recombinantBMP-4 and OP-1 (BMP-7) proteins are capable of inducing neural platecells to initiate dorsal neural cell fate differentiation (Liem et al.,Cell, 82, pp. 969-79 (1995)). At the spinal cord level, OP-1 and otherBMPs can induce neural crest cell differentiation. It is suggested thatOP-1 and these BMPs can induce many or all dorsal neural cell types,including roof plate cells, neural crest cells, and commissural neurons,depending on localized positional cues.

That several osteogenic proteins originally derived from bone matrixappear to be localized to embryonic nervous system and to haveneurogenic inductive properties makes it likely that these and othermembers of the BMP protein family will have additional tissue inductiveproperties that are not yet disclosed. It is envisioned that the abilityto enhance tissue inductive properties of morphogenic proteins using aMPSF as set forth herein will be useful for enhancing new tissueinductive properties of known morphogenic proteins. It is alsoenvisioned that the invention described herein will be useful forstimulating tissue inductive activities of new morphogenic proteins thatbelong to the BMP protein family as they are identified in the future.

Production of Morphogenic Proteins

The morphogenic proteins whose activity is enhanced in the presence of aMPSF according to this invention may be derived from a variety ofsources. Morphogenic proteins may be isolated from natural sources, ormay be produced by expressing an appropriate recombinant DNA molecule ina host cell. In addition, the morphogenic proteins of this invention maybe derived synthetically and synthetic morphogenic proteins mayoptionally be expressed from a recombinant DNA molecule in a host cell.

1. Naturally-derived morphogenic proteins

In one embodiment of this invention, morphogenic proteins are isolatedfrom natural sources and used in concert with a MPSF to induce tissueformation. Morphogenic proteins may be purified from tissue sources,preferably mammalian tissue sources, using conventional physical andchemical separation techniques well known to those of skill in the art.If a purification protocol is unpublished, as for a newly-identifiedmorphogenic protein for example, conventional protein purificationtechniques may be performed in combination with morphogenic activityassays following each step to trace the morphogenic activity through aseries of purification steps thereby establishing a viable purificationscheme. When available, immunological reagents may be used alone or inconjunction with the above techniques to purify morphogenic proteins.

This invention also provides native forms of osteogenic protein that actin concert with a MPSF to induce tissue formation. Osteogenic proteinmay be purified from natural sources according to protocols set forth,for example, in Oppermann et al., U.S. Pat. Nos. 5,324,819 and5,354,557, which are hereby incorporated by reference (see Example 1).

The osteogenic protein OP-1 has been described (see, e.g., Oppermann etal., U.S. Pat. No. 5,354,557). In its native form, OP-1 is glycosylatedand has an apparent molecular weight of about 30-35 kD as determined bySDS-PAGE. When reduced, the 30-35 kD protein gives rise to twoglycosylated polypeptide chains having apparent molecular weights thatmay range from about 15 kD to about 23 kD. In the reduced state, the30-35 kD protein has no detectable osteogenic activity. Thedeglycosylated protein, which has osteogenic activity, has an apparentmolecular weight of about 27 kD. When reduced, the 27 kD protein givesrise to the two deglycosylated polypeptides having molecular weights ofabout 14 kD to 16 kD.

The natural osteogenic proteins of this invention that act in concertwith a MPSF to induce tissue formation may include forms having varyingglycosylation patterns, varying N-termini, and active truncated ormutated forms of native protein.

2. Recombinantly-expressed Morphogenic Proteins

In another embodiment of this invention, a morphogenic protein isproduced by the expression of an appropriate recombinant DNA molecule ina host cell and is used in concert with a MPSF to induce tissueformation. The DNA and amino acid sequences of many BMPs and OPs havebeen reported, and methods for their recombinant production arepublished and otherwise known to those of skill in the art. For ageneral discussion of cloning and recombinant DNA technology, seeAusubel et al., supra; see also Watson et al., Recombinant DNA, 2d ed.1992 (W. H. Freeman and Co., New York).

The DNA sequences encoding bovine and human BMP-2 (formerly BMP-2A) andBMP-4 (formerly BMP-2B), and processes for recombinantly producing thecorresponding proteins are described in U.S. Pat. Nos. 5,011,691;5,013,649; 5,166,058 and 5,168,050.

The DNA and amino acid sequences of human and bovine BMP-5 and BMP-6,and methods for their recombinant production, are disclosed in U.S. Pat.No. 5,106,748, and U.S. Pat. No. 5,187,076, respectively; see also U.S.Pat. Nos. 5,011,691 and 5,344,654. Oppermann et al., U.S. Pat. Nos.5,011,691 and 5,258,494, disclose DNA and amino acid sequences encodingOP-1 (BMP-7), and methods for OP-1 recombinant expression. For analignment of BMP-2, BMP-4, BMP-5, BMP-6 and OP-1 (BMP-7) amino acidsequences, see WO 95/16034.

DNA sequences encoding BMP-8 are disclosed in WO 91/18098, and DNAsequences encoding BMP-9 in WO 93/00432. DNA and deduced amino acidsequences encoding BMP-10 and BMP-11 are disclosed in WO 94/26893, andWO 94/26892, respectively. DNA and deduced amino acid sequences forBMP-12 and BMP-13 are disclosed in WO 95/16035.

The above patent disclosures, which describe DNA and amino acidsequences, and methods for producing the BMPs and OPs encoded by thosesequences, are incorporated herein by reference.

To clone genes which encode new BMPs, OPs and other morphogenic proteinsidentified in extracts by bioassay, methods entailing "reverse genetics"may be employed. Such methods start with a protein of known or unknownfunction to obtain the gene which encodes that protein. Standard proteinpurification techniques may be used as an initial step in cloning thegene by reverse genetics. If enough protein can be purified to obtain apartial amino acid sequence, a degenerate DNA probe capable ofhybridizing to the DNA sequence that encodes that partial amino acidsequence may be designed, synthesized and used as a probe to isolatefull-length clones that encode that or a related morphogenic protein.

Alternatively, a partially-purified extract containing the morphogenicagent may be used to raise antibodies directed against that agent usingimmunological procedures well known in the art. Morphogenicprotein-specific antibodies may then be used as a probe to screenexpression libraries made from cDNAs (see, e.g., Broome and Gilbert,Proc. Natl. Acad. Sci. U.S.A., 75, pp. 2746-49 (1978; Young and Davis,Proc. Natl. Acad. Sci. U.S.A., 80, pp. 31-35 (1983)).

For cloning and expressing new BMPs, OPs and other morphogenic proteinsidentified based on DNA sequence homology, the homologous sequences maybe cloned and sequenced using standard recombinant DNA techniques. Withthe DNA sequence available, a DNA fragment encoding the morphogenicprotein may be inserted into an expression vector selected to work inconjunction with a desired host expression system. The DNA fragment iscloned into the vector such that its transcription is controlled by aheterologous promoter in the vector, preferably a promoter which may beoptionally regulated.

Some host-vector systems that are appropriate for the recombinantexpression of BMPs and OPs are disclosed in the references cited above.Useful host cells include but are not limited to bacteria such as E.coli, yeasts such as Saccharomyces and Picia, insect-baculovirus cellsystem, and primary, transformed or immortalized eukaryotic cells inculture. Preferred eukaryotic host cells include CHO, COS and BSC cells(see below).

An appropriate vector is selected according to the host system selected.Useful vectors include but are not limited to plasmids, cosmids,bacteriophage, insect and animal viral vectors, including retroviruses,and other single and double-stranded DNA viruses.

In one embodiment of this invention, the morphogenic protein used inconcert with a MPSF may be derived from a recombinant DNA moleculeexpressed in a prokaryotic host (Example 2A). Using recombinant DNAtechniques, various fusion genes have been constructed to inducerecombinant expression of naturally-sourced osteogenic sequences in E.coli (see, e.g., Oppermann et al., U.S. Pat. No. 5,354,557, incorporatedherein by reference). Using analogous procedures, DNAs comprisingtruncated forms of naturally-sourced morphogenic sequences may beprepared as fusion constructs linked by the acid labile cleavage site(Asp-Pro) to a leader sequence (such as the "MLE leader") suitable forpromoting expression in E. coli.

In another embodiment of this invention, the morphogenic protein used inconcert with a MPSF is expressed using a mammalian host/vector system(Example 2B). It may be preferable to recombinantly produce a mammalianprotein for therapeutic uses in mammalian cell culture systems in orderto produce a protein whose structure resembles more closely that of thenatural material. Recombinant protein production in mammalian cellsrequires the establishment of appropriate cells and cell lines that areeasy to transfect, are capable of stably maintaining foreign DNA with anunrearranged sequence, and which have the necessary cellular componentsfor efficient transcription, translation, post-translationalmodification and secretion of the protein. In addition, a suitablevector carrying the gene of interest is necessary.

DNA vector design for transfection into mammalian cells should includeappropriate sequences to promote expression of the gene of interest,including: appropriate transcription initiation, termination andenhancer sequences; efficient RNA processing signals such as splicingand polyadenylation signals; sequences that stabilize cytoplasmic mRNA;sequences that enhance translation efficiency (i.e., Kozak consensussequence); sequences that enhance protein stability; and when desired,sequences that enhance protein secretion.

Preferred DNA vectors also include a marker gene and means foramplifying the copy number of the gene of interest. DNA vectors may alsocomprise stabilizing sequences (e.g., ori- or ARS-like sequences andtelomere-like sequences), or may alternatively be designed to favordirected or non-directed integration into the host cell genome.

Substantial progress in the development of mammalian cell expressionsystems has been made in the last decade and many aspects of the systemare well characterized. A detailed review of the production of foreignproteins in mammalian cells, including useful cells, proteinexpression-promoting sequences, marker genes, and gene amplificationmethods, is disclosed in M. M. Bendig, Genetic Engineering, 7, pp.91-127 (1988).

Particular details of the transfection, expression and purification ofrecombinant proteins are well documented and are understood by those ofskill in the art. Further details on the various technical aspects ofeach of the steps used in recombinant production of foreign genes inmammalian cell expression systems can be found in a number of texts andlaboratory manuals in the art. See, e.g., F. M. Ausubel et al., ed.,Current Protocols in Molecular Biology, John Wiley & Sons, New York(1989).

Briefly, among the best characterized transcription promoters useful forexpressing a foreign gene in a particular mammalian cell are the SV40early promoter, the adenovirus major late promoter (AdMLP), the mousemetallothionein-I promoter (mMT-I), the Rous sarcoma virus (RSV) longterminal repeat (LTR), the mouse mammary tumor virus long terminalrepeat (MMTV-LTR), and the human cytomegalovirus majorintermediate-early promoter (hCMV). The DNA sequences for all of thesepromoters are known in the art and are available commercially.

One method of gene amplification in mammalian cell systems is the use ofthe selectable dihydrofolate reductase (DHFR) gene in a dhfr- cell line.Generally, the DHFR gene is provided on the vector carrying the gene ofinterest, and addition of increasing concentrations of the cytotoxicdrug methotrexate (MTX) leads to amplification of the DHFR gene copynumber, as well as that of the physically-associated gene of interest.DHFR as a selectable, amplifiable marker gene in transfected chinesehamster ovary cell lines (CHO cells) is particularly well characterizedin the art. Other useful amplifiable marker genes include the adenosinedeaminase (ADA) and glutamine synthetase (GS) genes.

In a preferred expression system, gene amplification is further enhancedby modifying marker gene expression regulatory sequences (e.g.,enhancer, promoter, and transcription or translation initiationsequences) to reduce the levels of marker protein produced. Lowering thelevel of DHFR transcription increases the DHFR gene copy number (and thephysically-associated gene) to enable the transfected cell to adapt togrowth in even low levels of methotrexate (e.g., 0.1 μM MTX). Preferredexpression vectors such as pH754 and pH752 (Oppermann et al., U.S. Pat.No. 5,354,557, FIGS. 19C and D), have been manipulated using standardrecombinant DNA technology, to create a weak DHFR promoter. As will beappreciated by those skilled in the art, other useful weak promoters,different from those disclosed and preferred herein, can be constructedusing standard vector construction methodologies. In addition, other,different regulatory sequences also can be modified to achieve the sameeffect.

Another gene amplification scheme relies on the temperature sensitivity(ts) of BSC40-tsA58 cells transfected with an SV40 vector. Temperaturereduction to 33° C. stabilizes the temperature sensitive SV40 T antigen,which leads to the excision and amplification of the integratedtransfected vector DNA thereby amplifying the physically associated geneof interest.

The choice of cells/cell lines is also important and depends on theneeds of the skilled practitioner. Monkey kidney cells (COS) providehigh levels of transient gene expression providing a useful means forrapidly testing vector construction and the expression of cloned genes.COS cells are transfected with a simian virus 40 (SV40) vector carryingthe gene of interest. The transfected COS cells eventually die, thuspreventing the long term production of the desired protein product.However, transient expression does not require the time consumingprocess required for the development of stable cell lines.

CHO cells are capable of successfully expressing a wide variety ofproteins from a broad range of cell types. Thus, while the glycosylationpattern on a recombinant protein produced in a mammalian cell expressionsystem may not be identical to the natural protein, the differences inoligosaccharide side chains are often not essential for biologicalactivity of the expressed protein.

Several different mammalian cell expression systems may be used toexpress recombinant morphogenic proteins to use in concert with a MPSFaccording to this invention. Stable cell lines have been developed usingCHO cells and a temperature-sensitive (ts) strain of BSC cells (simiankidney cells, BSC40-tsA58; Biotechnology, 6, pp. 1192-96 (1988)) for thelong term production of osteogenic protein OP-1. Among established celllines, CHO cells may be the best characterized to date, and are apreferred cell line for mammalian cell expression of recombinantmorphogenic proteins (Example 2b).

Two different promoters were found most useful to transcribe humanosteogenic protein sequences (hOP1; SEQ. ID No. 1): the CMV promoter andthe MMTV promoter, boosted by the enhancer sequence from the Roussarcoma virus LTR. The mMT promoter (mouse metallothionein promoter) andthe SV40 late promoter have also been tested. Several selection markergenes such as neo (neomycin) and DHFR are used.

Restriction maps and sources of various exemplary expression vectorsdesigned for OP-1 expression in mammalian cells have been described inOppermann et al., U.S. Pat. No. 5,354,557, incorporated herein byreference (see Example 2B). Each of these vector constructs employs afull-length human OP-1 cDNA sequence cloned into a conventional pUCvector (pUC-18).

It will be appreciated by those of skill in the art that DNA sequencesencoding truncated forms of osteogenic protein may also be used,provided that the expression vector or host cell then provides thesequences necessary to direct processing and secretion of the expressedprotein.

Recombinant OP-1 has been expressed in three different cell expressionsystems: COS cells for rapidly screening the functionality of thevarious expression vector constructs, CHO cells for the establishment ofstable cell lines, and BSC40-tsA58 cells as an alternative means ofproducing recombinant OP-1 protein. The CHO cell expression systemdisclosed herein is contemplated to be the best mode currently known forlong-term recombinant OP-1 production in mammalian cells (see Example2B).

As discussed above, several bone-derived osteogenic proteins (OPs) andBMPs are found as homo- and heterodimers comprising interchain disulfidebonds in their active forms. Methods for co-expressing and assemblingheteromeric polypeptide subunits in a host have been described (see,e.g., WO 93/09229, which is incorporated herein by reference). BMP-2,BMP-4, BMP-6 and BMP-7 (OP-1)--originally isolated from bone--arebioactive as either homodimers or heterodimers.

In addition, methods for making amino acid substitution mutations inBMPs and OPs that favor refolding and/or assembling subunits into formsthat exhibit greater morphogenic activity have also been described (U.S.Pat. No. 5,399,677, which is incorporated herein by reference).

Synthetic Non-native Morphogenic Proteins

In another embodiment of this invention, a morphogenic protein may beprepared synthetically for use in concert with a MPSF to induce tissueformation. Morphogenic proteins prepared synthetically may be native, ormay be non-native proteins, i.e., those not otherwise found in nature.

Non-native osteogenic proteins have been synthesized using a series ofconsensus DNA sequences (U.S. Pat. No. 5,324,819, incorporated herein byreference). These consensus sequences were designed based on partialamino acid sequence data obtained from natural osteogenic products andon their observed homologies with other genes reported in the literaturehaving a presumed or demonstrated developmental function.

Several of the biosynthetic consensus sequences (called consensusosteogenic proteins or "COPs") have been expressed as fusion proteins inprokaryotes. Purified fusion proteins may be cleaved, refolded, combinedwith at least one MPSF (optionally in a matrix or device), implanted inan established animal model and shown to have bone- and/orcartilage-inducing activity. The currently preferred syntheticosteogenic proteins comprise two synthetic amino acid sequencesdesignated COPS (Seq. ID No. 2) and COP7 (Seq. ID No. 3).

The amino acid sequences of these proteins are shown below, as set forthin Oppermann et al., U.S. Pat. Nos. 5,011,691 and 5,324,819, which areincorporated herein by reference:

    COP5      LYVDFS-DVGWDDWIVAPPGYQAFYCHGECPFPLAD                                           COP7 LYVDFS-DVGWNDWIVAPPGYHAFYCHGECPFPLAD                             - COP5 HFNSTN--H-AVVQTLVNSVNKI--PKACCVPTELSA                                 COP7 HLNSTN--H-AVVQTLVNSVNKI--PKACCVPTELSA                                     - COP5 ISMLYLDENEKVVLKYNQEMVVEGCGCR                                          COP7 ISMLYLDENEKVVLKYNQEMVVEGCGCR                                       

In these amino acid sequences, the dashes (-) are used as fillers onlyto line up comparable sequences in related proteins. Differences betweenthe aligned amino acid sequences are highlighted.

Thus in one embodiment of this invention, the morphogenic protein whichacts in concert with a MPSF to induce tissue formation is a syntheticosteogenic protein comprising a partial or the complete amino acidsequence of COP5 or COP7 such that it is capable of inducing tissueformation such as cartilage and/or bone formation in the presence of aMPSF when properly folded and implanted in a mammal.

COP proteins may be used in the presence of a MPSF to induce boneformation from osteoblasts when implanted in a favorable environment.Alternatively, COP proteins may be used in concert with a MPSF toproduce cartilage if implanted in an avascular locus or if an inhibitorto full bone development is implanted together with or present in thevicinity of the active morphogenic protein.

Preferably, the synthetic morphogenic protein which acts in concert witha MPSF of this invention comprises a protein which comprises a sequencesufficiently duplicative of the sequence of COP5 or COP7 such that it iscapable of tissue formation such as bone and/or cartilage formation whenproperly folded and implanted in a mammal in the presence of a MPSF.More preferably, the protein is less than about 200 amino acids long.

In one preferred embodiment, these synthetic proteins comprise speciesof the generic amino acid sequences:

     1       10        20        30        40        50                             CXXXXLXVXFXDXGWXXWXXXPXGXXAXYCXGXCXXPXXXXXXXXNHAXX                             -          60        70        80        90        100                       QXXVXXXNXXXXPXXCCXPXXXXXXXXLXXXXXXXVXLXXYXXMXVXXCXCX  ;                        - or                                                                          -  1       10        20        30        40        50                             LXVXFXDXGWXXWXXXPXGXXAXYCXGXCXXPXXXXXXXXNHAXX                             -          60        70        80        90        100                       QXXVXXXNXXXXPXXCCXPXXXXXXXXLXXXXXXXVXLXXYXXMXVXXCXCX                    

where the letters indicate the amino acid residues of standard singleletter code, and the Xs represent amino acid residues (residues 1-102and 5-102 of Seq. ID No. 4). Cysteine residues are highlighted.

Preferred amino acid sequences within the foregoing generic sequencesare:

    1        10        20        30        40        50                                  LYVDFRDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTNHAIV                                  K S S L  QE VIS E FD Y  E A AY MPESMKAS   VI                                  F E K I  DN     L    N  S   Q  ITK F P    TL                                      A    S      K                                                        -         60        70        80        90         100                       QTLVNSVNPGKIPKACCVPTELSAISMLYLDENENVVLKNYQDMVVEGCGCR                           SI HAI SEQV EP     EQMNSLAI FFNDQDK I RK EE T DA H H                             RF    T   S     K DPV V  Y N S     H RN   RS                                   N    S                      K       P     E                               - and                                                                         -  1       10        20        30        40        50                        CKRHPLYVDFRDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTNHAIV                             RRRS K S S L  QE VIS E FD Y  E A AY MPESMKAS   VI                               KE F E K I  DN     L    N  S   Q  ITK F P    TL                            Q         A    S      K                                                        -         60        70        80        90       100                         QTLVNSVNPGKIPKACCVPTELSAISMLYLDENENVVLKNYQDMVVEGCGCR                           SI HAI SEQV EP     EQMNSLAI FFNDQDK I RK RR T DA H H                             RF    T   S     K DPV V  Y N S     H RN   RS                                   N    S                      K       P     E                        

wherein each of the amino acids arranged vertically at each position inthe sequence may be used alternatively in various combinations (Seq. IDNo. 5). Note that these generic sequences have 6 and preferably 7cysteine residues where inter- or intramolecular disulfide bonds canform, and contain other critical amino acids which influence thetertiary structure of these osteogenic proteins.

Synthetic non-native osteogenic proteins may be chemically synthesizedor may be recombinantly expressed by introducing the synthetic DNAsequences on an expression vector into a host cell using proceduresdescribed above for recombinant expression of native protein sequences.These biosynthetic COP sequences are believed to dimerize duringrefolding, and appear not to be active when reduced. Homodimers orheterodimers may be assembled.

These and other synthetic non-native osteogenic proteins may be used inconcert with a MPSF and tested using in vitro, ex vivo or in vivobioassays for progenitor cell induction and tissue regenerationaccording to the procedures described herein. It is envisioned thatnon-native osteogenic protein/MPSF combinations will be capable ofinducing differentiation of certain neural lineages that can be inducedby native osteogenic proteins.

It is also envisioned that non-native osteogenic proteins in concertwith a MPSF will be capable of inducing other types of progenitor cellsto differentiate and proliferate. Thus non-native osteogenic protein andMPSF may be useful for the repair and regeneration not only of bone andcartilage tissue, but also of tendon, ligament, neural and potentiallyother types of tissue, and will thus be useful generally for tissuerepair and regeneration procedures.

Homologous Proteins Having Morphogenic Activity

The morphogenic proteins which act in concert with a MPSF to inducetissue formation according to this invention may be produced by therecombinant expression of DNA sequences isolated based on homology withthe osteogenic COP consensus sequences described above. Synthetic COPsequences such as those described above may be used as probes toretrieve related DNA sequences from a variety of species (see, e.g.,Oppermann et al., U.S. Pat. Nos. 5,011,591 and 5,258,494, which areincorporated herein by reference). COP sequences have retrieved genomicDNAs which were subsequently shown, when properly assembled, to encodeproteins which have true osteogenic activity, i.e., induce the fullcascade of events when properly implanted in a mammal leading to boneformation. Genomic DNAs encoding BMP-2 and OP-1 (BMP-7), for example,were isolated using this procedure.

Morphogenic proteins that are encoded by a gene which hybridizes with aCOP sequence probe are preferably assembled into a pair of subunitsdisulfide bonded to produce a dimeric species capable of inducing tissueformation when implanted in the presence of a MPSF into a mammal. Thedimeric species may comprise homo- or heterodimers of the COP-relatedpolypeptide assembled with a heterologous polypeptide. Recombinant formsof BMP-2 and BMP-4 have been shown to have cross-species osteogenicactivity as homodimers and as heterodimers assembled with OP-1 (BMP-7)subunits.

Morphogenic protein-encoding genes that hybridize to synthetic COPsequence probes include genes encoding Vg1, inhibin, DPP, OP-1(BMP-7),BMP-2 and BMP-4. Vg1 is a known Xenopus laevis morphogenic proteininvolved in early embryonic patterning. Inhibin is another developmentalgene that is a member of the BMP family of proteins from Xenopus laevis.DPP is an amino acid sequence encoded by a drosophila gene responsiblefor development of the dorso-ventral pattern. OP-1 (also called BMP-7),BMP-2 and BMP-4 are osteogenic proteins which can induce cartilage, boneand neural tissue formation (see below). Various combinations of thesepolypeptides, i.e., heterodimers and homodimers, have morphogenicactivity.

In another embodiment of this invention, a morphogenic protein that actsin concert with a MPSF may comprise a polypeptide encoded by a nucleicacid that can hybridize under stringent conditions to an "OPS" nucleicacid probe (Oppermann et al., U.S. Pat. No. 5,354,557). "OPS"--standingfor OP-1 "short"--refers to the portion of the human OP-1 proteindefining the conserved 6 cysteine skeleton in the C-terminal activeregion (97 amino acids; Seq. ID No. 1, residues 335-431).

One example of a stringent hybridization condition is hybridization in4×SSC at 65° C. (or 10° C. higher than the calculated meltingtemperature for a hybrid between the probe and a nucleic acid sequencecontaining no mis-matched base pairs), followed by washing in 0.1×SSC atthe hybridization temperature. Another stringent hybridization conditionis hybridization in 50% formamide, 4×SSC at 42° C. (see e.g., T.Maniatis et al., Molecular Cloning (A Laboratory Manual), Cold SpringHarbor Laboratory, pp.387-89 (1982)).

Thus, in view of this disclosure, the skilled practitioner may designand synthesize genes, or isolate genes from cDNA or genomic librarieswhich encode amino acid sequences associated with morphogenic activity.These genes can be expressed in prokaryotic or eukaryotic host cells toproduce large quantities of active osteogenic or otherwise morphogenicproteins. Recombinantly expressed proteins may be in native forms,truncated analogs, muteins, fusion proteins, and other constructed formscapable of inducing bone, cartilage, or other types of tissue formationas demonstrated by in vitro and ex vivo bioassays and in vivoimplantation in mammals, including humans.

Once the skilled practitioner has a bioassay that can detect one or moremorphogenic protein activities, a morphogenic protein stimulatory factor(MPSF) capable of stimulating that activity may be identified using thetechniques described herein.

Preferred Morphogenic Proteins

In one preferred embodiment of this invention, the morphogenic proteinwhose activity may be stimulated by the presence of a MPSF comprises apair of subunits disulfide bonded to produce a dimeric species, whereinat least one of the subunits comprises a recombinant polypeptidebelonging to the BMP protein family. The dimeric species may be ahomodimer or heterodimer and is capable of inducing cell proliferationand/or tissue formation when accessible to a progenitor cell in themammal. The progenitor cell may be induced to form one or more tissuetypes pereferably selected from the group consisting of endochondral orintramembranous bone, cartilage, tendon/ligament-like tissue, neuraltissue and other organ tissue types, including kidney tissue.

In another preferred embodiment, the morphogenic protein is anosteogenic protein that is capable of inducing the progenitor cell toform one or more tissue types selected from the group consisting ofendochondral or intramembranous bone and cartilage.

Preferred morphogenic and osteogenic proteins of this invention compriseat least one polypeptide selected from the group consisting of BMP-2,BMP-4, BMP-5, BMP-6, OP-1 (BMP-7), BMP-8, BMP-9, BMP-10, BMP-11, BMP-12,BMP-13, COP-5 and COP-7. Preferably, the morphogenic protein comprisesat least one polypeptide selected from the group consisting of OP-1(BMP-7), BMP-2, BMP-4 and BMP-6; more preferably, OP-1 (BMP-7)and BMP-2;and most preferably, OP-1 (BMP-7).

As the skilled practitioner will appreciate, the preferred morphogenicproteins of this invention whose activity is enhanced in the presence ofa MPSF will depend in part on the tissue type to be generated and on theselected implantation or treatment site. These variables may be testedempirically.

Morphogenic Protein Stimulatory Factors (MPSF)

A morphogenic protein stimulatory factor (MPSF) according to thisinvention is a factor that is capable of stimulating the ability of amorphogenic protein to induce tissue formation from a progenitor cell.In one embodiment of this invention, a method for improving the tissueinductive activity of a morphogenic protein in a mammal bycoadministering an effective amount of a MPSF is provided. The MPSF mayhave an additive effect on tissue induction by the morphogenic protein.Preferably, the MPSF has a synergistic effect on tissue induction by themorphogenic protein.

The progenitor cell that is induced to proliferate and/or differentiateby the morphogenic protein of this invention is preferably a mammaliancell. Preferred progenitor cells include mammalian chondroblasts,osteoblasts and neuroblasts, all earlier developmental precursorsthereof, and all cells that develop therefrom (e.g., chondroblasts,pre-chondroblasts and chondrocytes). However, morphogenic proteins arehighly conserved throughout evolution, and non-mammalian progenitorcells are also likely to be stimulated by same- or cross-speciesmorphogenic proteins and MPSF combinations. It is thus envisioned thatwhen schemes become available for implanting xenogeneic cells intohumans without causing adverse immunological reactions, non-mammalianprogenitor cells stimulated by morphogenic protein and a MPSF accordingto the procedures set forth herein will be useful for tissueregeneration and repair in humans.

One or more MPSFs are selected for use in concert with one or moremorphogenic proteins according to the desired tissue type to be inducedand the site at which the morphogenic protein and MPSF will beadministered. The particular choice of a morphogenic protein(s)/MPSF(s)combination and the relative concentrations at which they are combinedmay be varied systematically to optimize the tissue type induced at aselected treatment site using the procedures described herein.

The preferred morphogenic protein stimulatory factors (MPSFs) of thisinvention are selected from the group consisting of hormones, cytokinesand growth factors. Most preferred MPSFs for inducing bone and/orcartilage formation in concert with an osteogenic protein comprise atleast one compound selected from the group consisting of insulin-likegrowth factor I (IGF-I), estradiol, fibroblast growth factor (FGF),growth hormone (GH), growth and differentiation factor (GDF),hydrocortisone (HC), insulin, progesterone, parathyroid hormone (PTH),vitamin D (1,25-(OH)₂ D₃), retinoic acid and an interleukin,particularly IL-6. When the progenitor cell is an osteoblast stimulatedto form bone, preferred osteogenic protein/MPSF combinations excludeBMP-2 or BMP-3 homodimers used in concert with vitamin D or PTH.

In another preferred embodiment of this invention, the MPSF comprises acompound or an agent that is capable of increasing the bioactivity ofanother MPSF. Agents that increase MPSF bioactivity include, forexample, those that increase the synthesis, half-life, reactivity withother biomolecules such as binding proteins and receptors, or thebioavailability of the MPSF. These agents may comprise hormones, growthfactors, peptides, cytokines, carrier molecules such as proteins orlipids, or other factors that increase the expression or the stabilityof the MPSF.

For example, when the selected MPSF is IGF-I, agents that increase itsbioactivity include GH, PTH, vitamin D, and cAMP inducers, which maythus function as MPSFs according to this invention. In addition, almostall of the IGF-I in circulation and the extracellular space is bound bya group of high affinity binding proteins called IGFBPs which canaugment or inhibit IGF-I bioactivity (see, e.g., Jones and Clemmons,Endocrine Reviews, 16, pp. 3-34 (1995)). Thus IGFBPs and agents whichalter the levels of IGFBPs such that the bioactive IGF-I concentrationis ultimately increased will also function as a MPSF according to thisinvention.

These or other agents that increase IGF-I bioactivity may be used aloneas the primary MPSF, or one or more may be used as additional MPSFs incombination with IGF-I, to stimulate the tissue inductive activity ofthe morphogenic protein. One such preferred combination comprising atleast two MPSFs for cartilage and bone formation is osteogenic proteinOP-1, IGF-I and PTH (see below).

Preferably, the MPSF is present in an amount capable of synergisticallystimulating the tissue inductive activity of the morphogenic protein ina mammal. The relative concentrations of morphogenic protein and MPSFthat will optimally induce tissue formation when administered to amammal may be determined empirically by the skilled practitioner usingthe procedures described herein.

Testing Putative Morphogenic Protein Stimulatory Factors

To identify a MPSF that is capable of stimulating the tissue inductiveactivity of a chosen morphogenic protein, an appropriate assay must beselected. Initially, it is preferable to perform in vitro assays toidentify a MPSF that is capable of stimulating the tissue inductiveactivity of a morphogenic protein. A useful in vitro assay is one whichmonitors a nucleic acid or protein marker whose expression is known tocorrelate with the associated cell differentiation pathway.

Examples 3 and 4 describe experiments using the osteogenic protein OP-1to identify and to optimize an effective concentration of MPSF. Asdescribed above, OP-1 is known to have osteogenic and neurogenicactivity. Thus an in vitro assay looking at the expression of either anosteo- or neurogenic-associated marker in appropriately correspondingprogenitor cells can be used to identify one or more MPSFs that functionin concert with OP-1.

A preferred assay for testing potential MPSFs with OP-1 for osteogenicactivity is the alkaline phosphatase (AP) enzymatic assay. AP is anosteoblast differentiation marker in primary osteoblastic FRC (fetal ratcalvarial) cells. The OP-1-stimulated AP activity is the result ofincreased steady-state AP mRNA levels as measured by Northern analysis.The procedure is generally as follows.

First, a MPSF is identified by picking one or more concentrations of aMPSF and testing them alone or in the presence of a morphogenic protein(Examples 3 and 4). Second, the amount of MPSF required to achieveoptimal, preferably synergistic, tissue induction in concert with themorphogenic protein is determined by generating a dose response curve(Example 3).

Optionally, one or more additional MPSFs that stimulate or otherwisealter the morphogenic activity induced by a morphogenic protein and afirst MPSF may be identified and a new multi-factor dose response curvegenerated (Example 5).

Levels for additional biochemical markers for bone cell differentiationmay be measured to assay for synergistic effects of OP-1 and otherproteins belonging to the BMP family with IGF-I and other IGF-Iactivating agents. Other bone cell differentiation markers include butare not limited to: type I collagen, osteocalcin, osteopontin, bonesialoprotein and PTH-dependent cAMP levels.

FIG. 1 shows that IGF-I can act as a MPSF which stimulates theosteogenic activity of OP-1. Exogenous IGF-I elicits a stimulatoryeffect on the ability of OP-1 to induce FRC cell differentiation asmonitored by levels of cellular alkaline phosphatase (AP) activity.Exogenous IGF-I alone (up to 300 ng/ml) did not stimulate AP activity inFRC cells. However, IGF-I enhanced the OP-1-stimulated AP activity by3-4 fold. Thus the stimulatory effect of IGF-I is synergistic.

To show that the MPSF activity of IGF-I was not due to a contaminatingfactor present in the IGF-I preparation used in the above experiment, asimilar experiment was performed in the presence or absence of anIGF-I-specific antibody that blocks the action of IGF-I. As shown inFIG. 2, anti-IGFI antibody blocked, at least partially, theOP-1-stimulated alkaline phosphatase activity. Whereas OP-1 (500 ng/ml)stimulated AP activity by 1.6 fold above the vehicle-treated controlculture, co-incubation with anti-IGF-I antibody reduced the magnitude ofthe OP-1-induced stimulation about 50%. Increasing the amount ofantibody did not reduce the magnitude, suggesting that the amount ofantibody was not a limiting factor.

These results demonstrate that OP-1-induced differentiation ofosteoblastic cells may be stimulated by increasing IGF-I levels.

Once a morphogenic protein/MPSF pair has been identified, it isdesirable to identify the relative amounts of each component that arerequired to effectuate optimal levels of tissue inductive activity whenthe two components work in concert. This is done by assaying the tissueinductive activity produced when the concentration of each component issystematically varied independently from the other. The result of such astudy is a dose response curve for a given morphogenic protein/MPSFpair.

FIG. 3 shows the effect of varying IGF-I concentration (1-100 ng/ml) asa function of OP-1 concentration (0-500 ng/ml) on the synergisticenhancement of bone inducing activity. In the absence of OP-1, IGF-I didnot stimulate AP activity in FRC cells. However, at a OP-1 concentrationof 100 ng/ml, even a low concentration (10 ng/ml) of IGF-I potentiatedthe OP-1-stimulated AP activity by 1.5-2 fold. A maximum enhancement(about 2.5-fold) was observed at 25 ng/ml of IGF-I at an OP-1concentration of 200 ng/ml. IGF-I at higher concentrations no longerpotentiates the OP-1-stimulated AP activity. At these higher IGF-Iconcentrations, the OP-1-stimulated increase in AP activity is notinhibited.

Other factors still to be identified may also influence OP-1 inductiveactivity, and similar assays can be performed using OP-1 and IGF-I toidentify one or more additional MPSFs that can stimulate further theosteoinductive activity of OP-1 in the presence of IGF-I (Example 5).

To evaluate the effect of pre-treatment of FRC cells with OP-1 on thesynergistic effect of IGF-I, cells were first incubated in a constantconcentration of OP-1 (500 ng/ml). IGF-I (25 ng/ml) was added to theculture at different times subsequently, and the AP level was determinedat the end of 48 h of incubation. FIG. 4 shows that the maximumsynergistic effect was observed when FRC cells were treated with OP-1and IGF-I simultaneously. The effect was reduced significantly if IGF-Iwas added 6 h or later after OP-1 treatment. Pre-incubation of FRC cellswith IGF-I (25 ng/ml) for 24 h followed by OP-1 treatment (500 ng/ml)abolishes the synergistic effect. Thus when the morphogenic protein isOP-1 and the MPSF is IGF-I, it is preferred that they be administered ator at about the same time for the MPSF to have its maximum effect.

It may not hold true for every morphogenic protein/MPSF combination thatco-administration is optimal for inducing morphogenic activity. Forexample, when the MPSF (MPSF-1) is an agent that induces the expressionof another MPSF (MPSF-2), it may be found that pre-administering MPSF-1is preferred so that high levels of MPSF-2 are present when the selectedmorphogenic protein is administered. The procedures described herein canbe used by the skilled practitioner to optimize an administrationprotocol for a given morphogenic protein/MPSF combination to induce aselected tissue type at a selected treatment site.

The procedure described above for OP-1 and IGF-I may be used generallywith any selected morphogenic protein to test putative MPSFs compounds(Example 4). First, the morphogenic protein or agent is used to identifyand then to optimize conditions for an assay that accurately representsthe induction of a particular type of cell differentiation pathwayassociated with tissue formation. As described above, an in vitro assaythat is representative of the induction of the desired tissue type ispreferred at this stage. The assay may monitor mRNA or protein levels asa function of time or at a set time after administration of themorphogenic protein to cells or a tissue explant.

As described in Example 4, increasing concentrations of the followingcompounds were tested as MPSFs in combination with a singleconcentration (200 ng/ml) of osteogenic protein OP-1: a) estradiol (FIG.5); b) growth hormone (hGH; FIG. 6); c) hydrocortisone (HC; FIG. 7); d)insulin (FIG. 8); e) parathyroid hormone (PTH; FIG. 9); and f)progesterone (PG; FIG. 10). The results of these experiments demonstratethat each of the above compounds functions within a particularconcentration range as an MPSF in combination with OP-1.

In general, at least about 1 ng/ml of morphogenic protein is combinedwith at least about 0.01 ng/ml of MPSF to observe an increase in themorphogenic activity. Preferred concentration ranges for combinations ofosteogenic protein OP-1 and MPSF in inducing bone and cartilageformation, as determined in experiments such as those shown in FIGS. 3and 5-10, are shown in TABLE 1. It is envisioned that some of the MPSFs,particularly the hormones, may be more effective if alsopre-administered to the cells before the OP-1/MPSF composition isapplied.

                  TABLE 1                                                         ______________________________________                                        OP-1/MPSF Preferred Concentration Ranges                                             Morphogenic                                                              Protein (ng/ml)               MPSF                                          ______________________________________                                        OP-1   1-500         IGF-I       0.1-50 ng/ml                                   OP-1     1-500            estradiol  0.05-1000 nM                             OP-1     1-500            hGH         5.0-1000 ng/ml                          OP-1     1-500            HC         0.05-5.0 nM                              OP-1     1-500            insulin    0.01-1000 nM                             OP-1     1-500            PTH        10.0-1000 nM                             OP-1     1-500            PG         0.05-1000 nM                           ______________________________________                                    

Preferred concentration ranges for combinations of osteogenic proteinOP-1 and MPSF in inducing bone and cartilage formation are shown inTABLE 2.

                  TABLE 2                                                         ______________________________________                                        OP-1/MPSF More Preferred Concentrations                                              Morphogenic                                                              Protein  (ng/ml)   MPSF                                                     ______________________________________                                        OP-1   200           IGF-I      25 ng/ml                                        OP-1     200          estradiol  5 nM                                         OP-1     200          hGH        500-1000 ng/ml                               OP-1     200          HC         0.5-5.0 nM                                   OP-1     200          insulin    0.05 nM                                      OP-1     200          PTH        25-200 nM                                    OP-1     200          PG         0.05-5 nM                                  ______________________________________                                    

It will be appreciated by those skilled in the art that the preferredconcentration range of MPSF in a particular assay may vary depending onthe concentration of the morphogenic protein selected. Systematicvariation of the relative concentrations of the morphogenic protein andMPSF should thus be performed to optimize concentration ratios of thetwo factors. This may be done essentially as described in Example 2 andshown in FIG. 3 for OP-1 and IGF-I.

To determine whether other members of the IGF growth factor family alsoexhibit a synergistic effect with OP-1 similar to that observed forIGF-I, FRC cells were co-incubated with OP-1 (500 ng/ml) and varyingconcentrations of IGF-II. As shown in FIG. 11, IGF-II (10-300 ng/ml)neither enhanced nor inhibited OP-1-stimulated increase in AP activity.In addition, the level of AP activity in FRC cultures treated with IGF-I(25 ng/ml)+OP-1 (500 ng/ml) was similar to that in cultures treated withIGF-II (25 ng/ml)+IGF-I (25 ng/ml)+OP-1 (500 ng/ml). Thus IGF-II (925ng/ml) does not further potentiate the synergistic effect that IGF-I hason OP-1-induced tissue formation.

The data summarized in FIG. 12 indicate that TGF-β is not a MPSF incombination with OP-1 in the AP activity assay in FRC cells. TGF-β alonedid not stimulate AP activity. TGF-β (0.05-3.0 ng/ml) did not exhibitany synergistic effect with OP-1 on AP activity.

Based on morphogenic protein/MPSF dose response curves, compositionscomprising a morphogenic protein and a MPSF may be formulated at variousconcentration ratios and tested in a bioassay selected to represent thetissue inductive activity which will ultimately be used in the tissuetreatment. The preferred assay is ultimately an ex vivo or in vivotissue induction bioassay such as those described in Examples 7-13.

Pharmaceutical Compositions

The pharmaceutical compositions provided by this invention comprise atleast one and optionally more than one morphogenic protein/MPSFcombinations that are capable of inducing tissue formation whenadministered or implanted into a patient. The compositions of thisinvention will be administered at an effective dose to induce theparticular type of tissue at the treatment site selected according tothe particular clinical condition addressed. Determination of apreferred pharmaceutical formulation and a therapeutically efficientdose regiment for a given application is well within the skill of theart taking into consideration, for example, the administration mode, thecondition and weight of the patient, the extent of desired treatment andthe tolerance of the patient for the treatment.

Doses expected to be suitable starting points for optimizing treatmentregiments are based on the results of in vitro assays (e.g., Examples3-5), and ex vivo or in vivo assays (e.g., Examples 7-13). Based on theresults of such assays, a range of suitable morphogenic protein and MPSFconcentration ratios can be selected to test at a treatment site inanimals and then in humans.

Administration of the morphogenic proteins and MPSFs of this invention,including isolated and purified forms of morphogenic protein complexes,their salts or pharmaceutically acceptable derivatives thereof, may beaccomplished using any of the conventionally accepted modes ofadministration of agents which exhibit immunosuppressive activity.

The pharmaceutical compositions comprising a morphogenic protein and aMPSF of this invention may be in a variety of forms. These include, forexample, solid, semi-solid and liquid dosage forms such as tablets,pills, powders, liquid solutions or suspensions, suppositories, andinjectable and infusible solutions. The preferred form depends on theintended mode of administration and therapeutic application and may beselected by one skilled in the art. Modes of administration may includeoral, parenteral, subcutaneous, intravenous, intralesional or topicaladministration. In most cases, the pharmaceutical compositions of thisinvention will be administered in the vicinity of the treatment site inneed of tissue regeneration or repair.

The pharmaceutical compositions comprising a morphogenic protein and aMPSF of this invention may, for example, be placed into sterile,isotonic formulations with or without cofactors which stimulate uptakeor stability. The formulation is preferably liquid, or may belyophilized powder. For example, the morphogenic protein and MPSF ofthis invention may be diluted with a formulation buffer comprising 5.0mg/ml citric acid monohydrate, 2.7 mg/ml trisodium citrate, 41 mg/mlmannitol, 1 mg/ml glycine and 1 mg/ml polysorbate 20. This solution canbe lyophilized, stored under refrigeration and reconstituted prior toadministration with sterile Water-For-Injection (USP).

The compositions also will preferably include conventionalpharmaceutically acceptable carriers well known in the art (see forexample Remington's Pharmaceutical Sciences, 16th Edition, 1980, MacPublishing Company). Such pharmaceutically acceptable carriers mayinclude other medicinal agents, carriers, genetic carriers, adjuvants,excipients, etc., such as human serum albumin or plasma preparations.The compositions are preferably in the form of a unit dose and willusually be administered as a dose regiment that depends on theparticular tissue treatment.

The pharmaceutical compositions of this invention may also beadministered in conjunction with a morphogenic device using, forexample, microspheres, liposomes, other microparticulate deliverysystems or sustained release formulations placed in, near, or otherwisein communication with affected tissues or the bloodstream bathing thosetissues (see morphogenic devices, below).

Liposomes containing a morphogenic protein and a MPSF of this inventioncan be prepared by well-known methods (See, e.g. DE 3,218,121; Epsteinet al., Proc. Natl. Acad. Sci. U.S.A., 82, pp. 3688-92 (1985); Hwang etal., Proc. Natl. Acad. Sci. U.S.A., 77, pp. 4030-34 (1980); U.S. Pat.Nos. 4,485,045 and 4,544,545). Ordinarily the liposomes are of the small(about 200-800 Angstroms) unilamellar type in which the lipid content isgreater than about 30 mol. % cholesterol. The proportion of cholesterolis selected to control the optimal rate of morphogenic protein and MPSFrelease.

The morphogenic proteins and MPSFs of this invention may also beattached to liposomes containing other biologically active moleculessuch as immunosuppressive agents, cytokines, etc., to modulate the rateand characteristics of tissue induction. Attachment of morphogenicproteins and MPSFs to liposomes may be accomplished by any knowncross-linking agent such as heterobifunctional cross-linking agents thathave been widely used to couple toxins or chemotherapeutic agents toantibodies for targeted delivery. Conjugation to liposomes can also beaccomplished using the carbohydrate-directed cross-linking reagent4-(4-maleimidophenyl) butyric acid hydrazide (MPBH) (Duzgunes et al., J.Cell. Biochem. Abst. Suppl. 16E 77 (1992)).

Morphogenic Devices

The morphogenic devices of this invention comprise a morphogenic proteinand at least one MPSF dispersed in an implantable biocompatible carriermaterial that functions as a suitable delivery or support system for thecompounds. Suitable examples of sustained release carriers includesemipermeable polymer matrices in the form of shaped articles such assuppositories or capsules. Implantable or microcapsular sustainedrelease matrices include polylactides (U.S. Pat. No. 3,773,319; EP58,481), copolymers of L-glutamic acid and ethyl-L-glutamate (Sidman etal., Biopolymers, 22, pp. 547-56 (1985));poly(2-hydroxyethyl-methacrylate) or ethylene vinyl acetate (Langer etal., J. Biomed. Mater. Res., 15, pp. 167-277 (1981); Langer, Chem.Tech., 12, pp. 98-105 (1982)).

In one embodiment of this invention, the carrier of the morphogenicdevice comprises a biocompatible matrix made up of particles or porousmaterials. The pores are preferably of a dimension to permit progenitorcell migration and subsequent differentiation and proliferation. Variousmatrices known in the art can be employed (see, e.g., U.S. Pat. Nos.4,975,526; 5,162,114; 5,171,574 and WO 91/18558, which are hereinincorporated by reference).

The particle size should be within the range of 70 μm-850 μm, preferably70 μm-420 μm, most preferably 150 μm-420 μm. The matrix may befabricated by close packing particulate material into a shape spanningthe particular tissue defect to be treated. Alternatively, a materialthat is biocompatible, and preferably biodegradable in vivo may bestructured to serve as a temporary scaffold and substratum forrecruitment of migratory progenitor cells, and as a base for theirsubsequent anchoring and proliferation.

Useful matrix materials comprise, for example, collagen; homopolymers orcopolymers of glycolic acid, lactic acid, and butyric acid, includingderivatives thereof; and ceramics, such as hydroxyapatite, tricalciumphosphate and other calcium phosphates. Various combinations of these orother suitable matrix materials also may be useful as determined by theassays set forth herein.

Currently preferred carriers include particulate, demineralized,guanidine-extracted, species-specific (allogenic) bone, and speciallytreated particulate, protein-extracted, demineralized xenogenic bone(Example 6). Optionally, such xenogenic bone powder matrices also may betreated with proteases such as trypsin. Preferably, the xenogenicmatrices are treated with one or more fibril modifying agents toincrease the intraparticle intrusion volume (porosity) and surface area.Useful modifying agents include solvents such as dichloromethane,trichloroacetic acid, acetonitrile and acids such as trifluoroaceticacid and hydrogen fluoride. The currently preferred fibril-modifyingagent useful in formulating the matrices of this invention is a heatedaqueous medium, preferably an acidic aqueous medium having a pH lessthan about pH 4.5, most preferably having a pH within the range of aboutpH 2-pH 4. A currently preferred heated acidic aqueous medium is 0.1%acetic acid which has a pH of about 3. Heating demineralized,delipidated, guanidine-extracted bone collagen in an aqueous medium atelevated temperatures (e.g., in the range of about 37° C.-65° C.,preferably in the range of about 45° C.-60° C.) for approximately onehour generally is sufficient to achieve the desired surface morphology.Although the mechanism is not clear, it is hypothesized that the heattreatment alters the collagen fibrils, resulting in an increase in theparticle surface area.

Demineralized guanidine-extracted xenogenic bovine bone comprises amixture of additional materials that may be fractionated further usingstandard biomolecular purification techniques. For example,chromatographic separation of extract components followed by additionback to active matrix of the various extract fractions corresponding tothe chromatogram peaks may be used to improve matrix properties byfractionating away inhibitors of bone or tissue-inductive activity.

The matrix may also be substantially depleted in residual heavy metals.Treated as disclosed herein, individual heavy metal concentrations inthe matrix can be reduced to less than about 1 ppm.

One skilled in the art may create a biocompatible matrix of choicehaving a desired porosity or surface microtexture useful in theproduction of morphogenic devices to promote bone or other tissueinduction, or as a biodegradable sustained release implant. In addition,synthetically formulated matrices, prepared as disclosed herein, may beused.

General Consideration of Matrix Properties

The currently preferred carrier material is a xenogenic bone-derivedparticulate matrix treated as described herein. This carrier may bereplaced by either a biodegradable-synthetic or a synthetic-inorganicmatrix (e.g., hydroxyapatite (HAP), collagen, carboxymethyl-cellulose,tricalcium phosphate or polylactic acid, polyglycolic acid, polybutyricacid and various copolymers thereof.)

Matrix geometry, particle size, the presence of surface charge, and thedegree of both intra- and inter-particle porosity are all important tosuccessful matrix performance. Studies have shown that surface charge,particle size, the presence of mineral, and the methodology forcombining matrix and morphogenic proteins all play a role in achievingsuccessful tissue induction.

For example, in bone formation using osteogenic protein OP-1 and a MPSF,perturbation of the matrix charge by chemical modification can abolishbone inductive responses. Particle size influences the quantitativeresponse of new bone; particles between 70 μm and 420 μm elicit themaximum response. Contamination of the matrix with bone mineral willinhibit bone formation. Most importantly, the procedures used toformulate osteogenic protein and MPSF onto the matrix are extremelysensitive to the physical and chemical state of both the proteins andthe matrix.

The sequential cellular reactions in the interface of the bonematrix/osteogenic protein implants are complex. The multistep cascadeincludes: binding of fibrin and fibronectin to implanted matrix,migration and proliferation of mesenchymal cells, differentiation of theprogenitor cells into chondroblasts, cartilage formation, cartilagecalcification, vascular invasion, bone formation, remodeling, and bonemarrow differentiation.

A successful carrier for morphogenic protein and MPSF should performseveral important functions. It should act as a slow release deliverysystem of morphogenic protein and MPSF, protect the morphogenic proteinand MPSF from non-specific proteolysis, and should accommodate each stepof the cellular responses involved in progenitor cell induction duringtissue development.

In addition, selected materials must be biocompatible in vivo andpreferably biodegradable; the carrier preferably acts as a temporaryscaffold until replaced completely by new bone or tissue. Polylacticacid (PLA), polyglycolic acid (PGA), and various combinations havedifferent dissolution rates in vivo. In bones, the dissolution rates canvary according to whether the implant is placed in cortical ortrabecular bone.

The preferred osteogenic device matrix material, prepared from xenogenicbone and treated as disclosed herein, produces an implantable materialuseful in a variety of clinical settings. In addition to its use as amatrix for bone formation in various orthopedic, periodontal, andreconstructive procedures, the matrix also may be used as a sustainedrelease carrier, or as a collagenous coating for orthopedic or generalprosthetic implants.

The matrix may be shaped as desired in anticipation of surgery or shapedby the physician or technician during surgery. It is preferred to shapethe matrix to span a tissue defect and to take the desired form of thenew tissue. In the case of bone repair of a non-union defect, forexample, it is desirable to use dimensions that span the non-union. Ratstudies show that the new bone is formed essentially having thedimensions of the device implanted. Thus, the material may be used fortopical, subcutaneous, intraperitoneal, or intramuscular implants. Inbone formation procedures, the material is slowly absorbed by the bodyand is replaced by bone in the shape of or very nearly the shape of theimplant.

The matrix may comprise a shape-retaining solid made of loosely-adheredparticulate material, e.g., collagen. It may also comprise a molded,porous solid, or simply an aggregation of close-packed particles held inplace by surrounding tissue. Masticated muscle or other tissue may alsobe used. Large allogenic bone implants can act as a carrier for thematrix if their marrow cavities are cleaned and packed with particlescomprising dispersed osteogenic protein and MPSF. The matrix may alsotake the form of a paste or a hydrogel.

When the carrier material comprises a hydrogel matrix, it refers to athree dimensional network of cross-linked hydrophilic polymers in theform of a gel substantially composed of water, preferably but notlimited to gels being greater than 90% water. Hydrogel matrices cancarry a net positive or net negative charge, or may be neutral. Atypical net negative charged matrix is alginate. Hydrogels carrying anet positive charge may be typified by extracellular matrix componentssuch as collagen and laminin. Examples of commercially availableextracellular matrix components include Matrigel™ and Vitrogen™. Anexample of a net neutral hydrogel is highly crosslinked polyethyleneoxide, or polyvinyalcohol.

Various growth factors, cytokines, hormones, trophic agents andtherapeutic compositions including antibiotics and chemotherapeuticagents, enzymes, enzyme inhibitors and other bioactive agents also maybe adsorbed onto or dispersed within the carrier material comprising themorphogenic protein and MPSF, and will also be released over time at theimplantation site as the matrix material is slowly absorbed.

Other Tissue-Specific Matrices

In addition to the naturally-derived bone matrices described above,useful matrices may also be formulated synthetically by adding togetherreagents that have been appropriately modified. One example of such amatrix is the porous, biocompatible, in vivo biodegradable syntheticmatrix disclosed in WO91/18558, the disclosure of which is herebyincorporated by reference.

Briefly, the matrix comprises a porous crosslinked structural polymer ofbiocompatible, biodegradable collagen, most preferably tissue-specificcollagen, and appropriate, tissue-specific glycosaminoglycans astissue-specific cell attachment factors. Bone tissue-specific collagen(e.g., Type I collagen) derived from a number of sources may be suitablefor use in these synthetic matrices, including soluble collagen,acid-soluble collagen, collagen soluble in neutral or basic aqueoussolutions, as well as those collagens which are commercially available.In addition, Type II collagen, as found in cartilage, also may be usedin combination with Type I collagen.

Glycosaminoglycans (GAGs) or mucopolysaccharides are polysaccharidesmade up of residues of hexoamines glycosidically bound and alternatingin a more-or-less regular manner with either hexouronic acid or hexosemoieties. GAGs are of animal origin and have a tissue specificdistribution (see, e.g., Dodgson et al., in Carbohydrate Metabolism andits Disorders, Dickens et al., eds., Vol. 1, Academic Press (1968)).Reaction with the GAGs also provides collagen with another valuableproperty, i.e., inability to provoke an immune reaction (foreign bodyreaction) from an animal host.

Useful GAGs include those containing sulfate groups, such as hyaluronicacid, heparin, heparin sulfate, chondroitin 6-sulfate, chondroitin4-sulfate, dermatan sulfate, and keratin sulfate. For osteogenicdevices, chondroitin 6-sulfate currently is preferred. Other GAGs alsomay be suitable for forming the matrix described herein, and thoseskilled in the art will either know or be able to ascertain othersuitable GAGs using no more than routine experimentation. For a moredetailed description of mucopolysaccharides, see Aspinall,Polysaccharides, Pergamon Press, Oxford (1970).

Collagen can be reacted with a GAG in aqueous acidic solutions,preferably in diluted acetic acid solutions. By adding the GAG dropwiseinto the aqueous collagen dispersion, coprecipitates of tangled collagenfibrils coated with GAG results. This tangled mass of fibers then can behomogenized to form a homogeneous dispersion of fine fibers and thenfiltered and dried.

Insolubility of the collagen-GAG products can be raised to the desireddegree by covalently cross-linking these materials, which also serves toraise the resistance to resorption of these materials. In general, anycovalent G60 cross-linking method suitable for cross-linking collagenalso is suitable for cross-linking these composite materials, althoughcross-linking by a dehydrothermal process is preferred.

When dry, the cross-linked particles are essentially spherical withdiameters of about 500 μm. Scanning electron microscopy shows pores ofabout 20 μm on the surface and 40 μm on the interior. The interior ismade up of both fibrous and sheet-like structures, providing surfacesfor cell attachment. The voids interconnect, providing access to thecells throughout the interior of the particle. The material appears tobe roughly 99.5% void volume, making the material very efficient interms of the potential cell mass that can be grown per gram ofmicrocarrier.

Another useful synthetic matrix is one formulated from biocompatible, invivo biodegradable synthetic polymers, such as those composed ofglycolic acid, lactic acid and/or butyric acid, including copolymers andderivatives thereof. These polymers are well described in the art andare available commercially. For example, polymers composed of polylacticacid (e.g., MW 100 ka), 80% polylactide/20% glycoside or poly3-hydroxybutyric acid (e.g., MW 30 ka) all may be purchased fromPolySciences, Inc. The polymer compositions generally are obtained inparticulate form and the morphogenic devices preferably fabricated undernonaqueous conditions (e.g., in an ethanol-trifluoroacetic acidsolution, EtOH/TFA) to avoid hydrolysis of the polymers. In addition,one can alter the morphology of the particulate polymer compositions,for example to increase porosity, using any of a number of particularsolvent treatments known in the art.

Fabrication of morphogenic device

The naturally-sourced, synthetic and recombinant morphogenic proteinsand MPSFs as set forth above, as well as other constructs, can becombined and dispersed in a suitable matrix preparation using any of themethods described. In general, about 500-1000 ng of active morphogenicprotein and about 10-200 ng of an active MPSF are combined with 25 mg ofthe inactive carrier matrix for rat bioassays. In larger animals,typically about 0.8-1 mg of active morphogenic protein per gram ofcarrier is combined with 100 ng or more of an active MPSF. The optimalratios of morphogenic protein to MPSF for a specific combination andtissue type may be determined empirically by those of skill in the artaccording to the procedures set forth herein. Greater amounts may beused for large implants.

Prosthetic Devices

In another embodiment of this invention, an implantable prostheticdevice comprising an osteogenic protein and a MPSF is provided. Anyprosthetic implant selected for a particular treatment by the skilledpractitioner may be used in combination with a composition comprising atleast one osteogenic protein and at least one MPSF according to thisinvention. The prosthesis may be made from a material comprising metalor ceramic. Preferred prosthetic devices are selected from the groupconsisting of a hip device, a screw, a rod and a titanium cage for spinefusion.

The osteogenic composition is disposed on the prosthetic implant on asurface region that is implantable adjacent to a target tissue in themammal. Preferably, the mammal is a human patient. The composition isdisposed on the surface of the implant in an amount sufficient topromote enhanced tissue growth into the surface. The amount of thecomposition sufficient to promote enhanced tissue growth may bedetermined empirically by those of skill in the art using bioassays suchas those described herein and in Rueger et al., U.S. Pat. No. 5,344,654,which is incorporated herein by reference. Preferably, animal studiesare performed to optimize the concentration of the compositioncomponents before a similar prosthetic device is used in the humanpatient. Such prosthetic devices will be useful for repairing orthopedicdefects, injuries or anomalies in the treated mammal.

Thus this invention also provides a method for promoting in vivointegration of an implantable prosthetic device into a target tissue ofa mammal comprising the steps of providing on a surface of theprosthetic device a composition comprising at least one osteogenicprotein and at least one MPSF, and implanting the device in a mammal ata locus where the target tissue and the surface of the prosthetic deviceare maintained at least partially in contact for a time sufficient topermit enhanced tissue growth between the target tissue and the device.

Bioassays

The various morphogenic compositions and devices of this invention arepreferably evaluated with ex vivo or in vivo bioassays. Studies in ratsshow the osteogenic effect in an appropriate matrix to be dependent onthe dose of morphogenic protein dispersed in the matrix. No activity isobserved if the matrix is implanted alone. In vivo bioassays performedin the rat model also have shown that demineralized, guanidine-extracted xenogenic bone matrix materials of the type described in theliterature generally are ineffective as a carrier, can fail to inducebone, and can produce an inflammatory and immunological response whenimplanted unless treated as disclosed above. In certain species (e.g.,monkey), allogenic matrix materials also apparently are ineffective ascarriers (Aspenberg et al., J. Bone Joint Surgery, 70, pp. 625-627(1988)). Examples 6-13 set forth various procedures for preparingmorphogenic devices and for evaluating their morphogenic utility usingin vivo mammalian bioassays.

A rat bioassay for bone induction--based on the bioassay for inductionof bone differentiation activity as described by Sampath and Reddi(Proc. Natl. Acad. Sci. USA, 80, pp. 6591-95 (1983)), hereinincorporated by reference--may be used to monitor osteogenic activity ofosteogenic proteins in concert with one or more MPSFs (Example 7). Ratbioassays are preferred as the first step in moving from in vitro assayresults to in vivo implantation studies.

The feline and rabbit as established large animal efficacy models forosteogenic device testing have been described in detail (Oppermann etal., U.S. Pat. No. 5,354,557; Example 8 and Example 9). The felinefemoral model, the rabbit ulnar model, the dog ulnar model (Example 10)or the monkey model (Example 11) are all useful assays to evaluatewhether the compositions and devices of this invention comprising one ormore osteogenic proteins in combination with one or more MPSFs canenhance bone regeneration in vivo and for determining optimal dosing ofmorphogenic protein/MPSF combinations.

Preferably, results from the rat bioassay (Example 7) are used as astarting point for optimization studies in one of these larger animalmodels. Most preferably, the larger animal study is performed in the dogor the monkey.

While the feline and the rabbit studies use allogenic matrices asosteogenic device carrier material, appropriate treatment as describedherein of any bone-derived or synthetic matrix material is anticipatedto render the matrix suitable for xenogenic implants. However, resultsin the rabbit tend to be less predictable when using osteogenic proteins(with or without MPSFs) dispersed in bovine-derived collagen matrix.

Recombinant BMP-2 is effective in repairing large bone defects in avariety of other mammalian bioassay models. Implanted osteogenic devicescomprising BMP-2 successfully heal segmental defects in rat femurs(Yasko et al., J. Bone Joint Sura., 74A, pp. 659-70 (1992), sheep femurs(Gerhart et al., Clin. Orthop., 293, pp. 317-26 (1993), in caninemandibles (Toriumi et al., Arch. Otolaryngol. Head Neck Surg., 117, pp.1101-12 (1991), and in skull defects in rats and dogs.

The procedures described above may be used to assess the ability of oneor more MPSFs to enhance the osteogenic activity of one or moreosteogenic proteins in bone and/or cartilage regeneration and repair invivo. These procedures may also be used to optimize conditions forenhancing osteogenic activity using one or more MPSFs. It is anticipatedthat the efficacy of any osteogenic protein/MPSF combination may becharacterized using these assays. Various osteogenic protein/MPSFcombinations, dose-response curves, various naturally-derived orsynthetic matrices, and any other desired variations on the osteogenicdevice components may be tested using the procedures essentially asdescribed.

Tendon/ligament-like tissue formation bioassay

A modified version of the Sampath and Reddi rat ectopic implant assay(see above) has been reported by Celeste et al., WO 95/16035, which ishereby incorporated by reference. The modified assay monitors tendon andligament-like tissue formation induced by morphogenic proteins (such asBMP-12, BMP-13 and human MP52). This tendon/ligament-like tissue assaymay be used to identify MPSFs that stimulate tendon/ligament-like tissueformation by BMP-12, BMP-13 or other morphogenic proteins in aparticular treatment site (Example 12). The assay may also be used tooptimize concentrations and treatment schedules for therapeutic tissuerepair regiments.

It should be understood that the above experimental procedure may bemodified within the skill of the art in a number of ways to be useful indetermining whether a morphogenic device is capable of inducing tendonand/or ligament-like tissue in vivo. It may be used to test variouscombinations of morphogenic protein/MPSF combinations, and to produce anin vivo dose response curve useful in determining effective relativeconcentrations of morphogenic proteins and MPSFs. It may also be usedfor identifying concentration ranges in which a particular MPSF mayadditively or synergistically enhance the inductive activity of aparticular morphogenic protein.

The osteogenic proteins BMP-4 and BMP-7 (OP-1) can induce ventral neuralplate explants to undergo differentiation into dorsal neural cell fates(Liem et al., Cell, 82, pp. 969-79 (1995)). Molecular markers of dorsalcell differentiation are described in Liem et al. These markers includePAX3 and MSX, whose expression delineates an early stage of neural platecell differentiation; DSL-1 (a BMP-like molecule) delineatingdifferentiation of dorsal neural plate cells at a stage after neuraltube closure; and SLUG protein, whose expression after neural tubeclosure defines premigratory neural crest cells. Expression of thesedorsal markers can be induced in ventral neural plate explants byectopic BMP4 and BMP-7 (OP-1).

A peripheral nerve regeneration assay using the morphogenic proteinBMP-2 has been described (Wang et al., WO 95/05846, which is herebyincorporated by reference). The assay involves the implantation ofneurogenic devices in the vicinity of severed sciatic nerves in rats.This procedure may be used to assess the ability of a putative MPSF tostimulate the neuronal inducing activity of homo- and heterodimers ofmorphogenic proteins having neurogenic activity, such as BMP-2, BMP-4,BMP-6 and OP-1 (BMP-7), or of any other selected neurogenic protein/MPSFcombinations (Example 13).

Utility of Morphogenic Compositions and Devices

The morphogenic compositions and devices comprising a morphogenicprotein and MPSF disclosed herein will permit the physician to treat avariety of tissue injuries, tissue degenerative or disease conditionsand disorders that can be ameliorated or remedied by localized,stimulated tissue regeneration or repair.

The morphogenic devices of this invention may be used to induce localtissue formation from a progenitor cell in a mammal by implanting thedevice at a locus accessible to at least one progenitor cell of themammal. The morphogenic devices of this invention may be used alone orin combination with other therapies for tissue repair and regeneration.

The morphogenic devices of this invention may also be implanted in orsurrounding a joint for use in cartilage and soft tissue repair, or inor surrounding nervous system-associated tissue for use in neuralregeneration and repair.

The tissue specificity of the particular morphogenic protein--orcombination of morphogenic proteins with other biological factors--willdetermine the cell types or tissues that will be amenable to suchtreatments and can be selected by one skilled in the art. The ability toenhance morphogenic protein-induced tissue regeneration byco-administering a MPSF according to the present invention is thus notbelieved to be limited to any particular cell-type or tissue. It isenvisioned that the invention as disclosed herein can be practiced toenhance the activities of new morphogenic proteins and to enhance newtissue inductive functions as they are discovered in the future.

The osteogenic compositions and devices comprising an osteogenic proteinand a MPSF will permit the physician to obtain predictable bone and/orcartilage formation using less osteogenic protein to achieve at leastabout the same extent of bone or cartilage formation. The osteogeniccompositions and devices of this invention may be used to treat moreefficiently and/or effectively all of the injuries, anomalies anddisorders that have been described in the prior art of osteogenicdevices. These include, for example, forming local bone in fractures,non-union fractures, fusions and bony voids such as those created intumor resections or those resulting from cysts; treating acquired andcongenital craniofacial and other skeletal or dental anomalies (seee.g., Glowacki et al., Lancet, 1, pp. 959-63 (1981)); performing dentaland periodontal reconstructions where lost bone replacement or boneaugmentation is required such as in a jaw bone; and supplementingalveolar bone loss resulting from periodontal disease to delay orprevent tooth loss (see e.g., Sigurdsson et al., J. Periodontol., 66,pp. 511-21 (1995)).

An osteogenic device of this invention which comprises a matrixcomprising allogenic bone may also be implanted at a site in need ofbone replacement to accelerate allograft repair and incorporation in amammal.

Another potential clinical application of the improved osteogenicdevices of this invention is in cartilage repair, for example, followingjoint injury or in the treatment of osteoarthritis. The ability toenhance the cartilage-inducing activity of morphogenic proteins byco-administering a MPSF may permit faster or more extensive tissuerepair and replacement using the same or lower levels of morphogenicproteins.

The morphogenic compositions and devices of this invention will beuseful in treating certain congenital diseases and developmentalabnormalities of cartilage, bone and other tissues. For example,homozygous OP-1 (BMP-7)-deficient mice die within 24 hours after birthdue to kidney failure (Luo et al., J. Bone Min. Res., 10 (Supp. 1), pp.S163 (1995)). Kidney failure in these mice is associated with thefailure to form renal glomeruli due to lack of mesenchymal tissuecondensation. OP-1-deficient mice also have various skeletalabnormalities associated with their hindlimbs, rib cage and skull, arepolydactyl, and exhibit aberrant retinal development. These results, incombination with those discussed above concerning the ability of OP-1 toinduce differentiation into dorsal neural cell fates, indicate that OP-1plays an important role in epithelial-mesenchymal interactions duringdevelopment. It is anticipated that the compositions, devices andmethods of this invention may be useful in the future for amelioratingthese and other developmental abnormalities.

Developmental abnormalities of the bone may affect isolated or multipleregions of the skeleton or of a particular supportive or connectivetissue type. These abnormalities often require complicated bonetransplantation procedures and orthopedic devices. The tissue repair andregeneration required after such procedures may occur more quickly andcompletely with the use of morphogenic proteins used in combination withMPSFs according to this invention.

Examples of heritable conditions, including congenital bone diseases,for which use of the morphogenic compositions and devices of thisinvention will be useful include osteogenesis imperfecta, the Hurler andMarfan syndromes, and several disorders of epiphyseal and metaphysealgrowth centers such as is presented in hypophosphatasia, a deficiency inalkaline phosphatase enzymatic activity.

Inflammatory joint diseases may also benefit from the improvedmorphogenic compositions and devices of this invention. These includebut are not limited to infectious, non-infectious, rheumatoid andpsoriatic arthritis, bursitis, ulcerative colitis, regional enteritis,Whipple's disease, and ankylosing spondylitis (also called MarieStrumpell or Bechterew's disease); the so-called "collagen diseases"such as systemic lupus erythematosus (SLE), progressive systemicsclerosis (scleroderma), polymyositis (dermatomyositis), necrotizingvasculitides, Sjogren's syndrome (sicca syndrome), rheumatic fever,amyloidosis, thrombotic thrombocytopenic purpura and relapsingpolychondritis. Heritable disorders of connective tissue includeMarfan's syndrome, homocystinuria, Ehlers-Danlos syndrome, osteogenesisimperfecta, alkaptonuria, pseudoxanthoma elasticum, cutis laxa, Hurler'ssyndrome, and myositis ossificans progressiva.

The following are examples which illustrate the morphogenic compositionsand devices of this invention, and methods used to characterize them.These examples should not be construed as limiting: the examples areincluded for purposes of illustration and the present invention islimited only by the claims.

EXAMPLE 1 Preparation of OP-1 from Natural Sources

For a detailed description of the procedure for purifying OP-1 frombovine bone, see Oppermann et al., U.S. Pat. No. 5,324,819, which isincorporated herein by reference.

Preparation of Demineralized Bone

Demineralized bovine bone matrix is prepared using previously publishedprocedures (Sampath and Reddi, Proc. Natl. Acad. Sci. USA, 80, pp.6591-95 (1983)). Fresh bovine diaphyseal bones (age 1-10 days) arestripped of muscle and fat, cleaned of periosteum, demarrowed bypressure with cold water, dipped in cold absolute ethanol, and stored at-20° C. They are then dried and fragmented by crushing and pulverized ina large mill using liquid nitrogen to prevent heating. The pulverizedbone is milled to a particle size between 70-420 mm and is defatted bytwo washes of approximately two hours duration with three volumes ofchloroform and methanol (3:1). The particulate bone is then washed withone volume of absolute ethanol and dried over one volume of anhydrousether. Alternatively, Bovine Cortical Bone Powder (75-425 mm) may bepurchased from American Biomaterials.

The defatted bone powder is demineralized with 10 volumes of 0.5 N HClat 4° C. for 40 min., four times. Finally, neutralizing washes are doneon the demineralized bone powder with a large volume of water.

Demineralized bone powder is then used as a starting material forperforming the following purification steps, which are explained indetail in Oppermann et al., U.S. Pat. No. 5,324,819:

1. Dissociative extraction and ethanol precipitation;

2. Heparin-sepharose chromatography I;

3. Hydroxyapatite-ultrogel chromatography;

4. Sephacryl S-300 gel exclusion chromatography;

5. Heparin-sepharose chromatography II; and

6. Reverse phase HPLC

SDS gel electrophoresis may be performed to visualize and characterizefurther the species separated by HPLC; gel eluted species may befiltered, concentrated and prepared further for sequencing and otherdesired characterizations. The yield is typically 0.5 to 1.0 μgsubstantially pure osteogenic protein per kg of bone.

For additional details on these procedures and the chemicalcharacterization of the naturally-derived osteogenic proteins, see alsoOppermann et al., U.S. Pat. No. 5,258,494, which is incorporated hereinby reference.

EXAMPLE 2 Preparation of Recombinant Osteogenic Protein

A. Expression in E. Coli

Using recombinant DNA techniques, various fusion genes can beconstructed to induce recombinant expression of naturally-sourcedosteogenic sequences in a procaryotic host such as E. coli. Full-lengthor truncated forms of the morphogenic genes encoding OP-1 or BMP-2 werecloned into a bacterial expression vector downstream from an acid labileAsp-Pro cleavage site under the control of a synthetic trppromoter-operator. Vectors were introduced into an appropriate E. colistrain by transformation and the bacteria were grown up to produceinsoluble inclusion bodies.

The inclusion bodies were solubilized in 8M urea following lysis,dialyzed against 1% acetic acid, and partly purified by differentialsolubilization. Constructs containing the Asp-Pro site were cleaved withacid. The resulting products were passed through a Sephacryl-200HR or SPTrisacyl column to further purify the proteins, and then subjected toHPLC on a semi-prep C-18 column to separate the leader proteins andother minor impurities from the morphogenic protein constructs.

Morphogenic proteins OP-1 and BMP-2 were purified by chromatography onheparin-Sepharose. The output of the HPLC column was lyophilized at pH 2so that it remained reduced.

Conditions for refolding were at pH 8.0 using Tris buffer and 6Mguanidine-HCl at a protein concentration of several mg/ml. Thosesolutions were diluted with water to produce a 2M or 3M guanidineconcentration and left for 18 hours at 4° C. Air dissolved or entrainedin the buffer assured oxidation of the protein in these circumstances.

Samples of the various purified constructs and various mixtures of pairsof the constructs refolded together were applied to SDS polyacrylamidegels, separated by electrophoresis, sliced, incorporated in a matrix asdisclosed below, and tested for osteogenic activity.

These studies demonstrated that each of the constructs (full-length ortruncated versions) have true osteogenic activity. In addition, mixedspecies including heterodimers were also osteogenically active and mayinclude heterodimers. For specific combinations tested, see Oppermann etal., U.S. Pat. No. 5,354,557). Finally, single and mixed species ofanalogs of the active region, e.g., COP5 and COP7, disclosed in U.S.Pat. No. 5,011,691, also induce osteogenesis, as determined byhistological examination.

After N-terminal sequencing of the various constructs to confirm theiridentity, polyclonal antisera against the recombinant presumed matureform proteins were produced. The human OP-1 antisera reacted with boththe glycosylated and unglycosylated higher molecular weight subunits ofnaturally sourced bovine material. Antisera against recombinant maturehuman BMP-2 reacted with both the glycosylated and unglycosylated lowermolecular weight subunit of naturally sourced bovine material. Whilethere was some cross-reactivity, this was expected in view of thesignificant homology between BMP-2 and OP-1 (approx. 60% identity), andthe likelihood that degraded OP-1 generated during purificationcontaminates the lower molecular weight subunit. Both antisera reactwith the naturally sourced 30 ka dimeric bOP.

In addition, synthetic osteogenic sequences produced by assembly ofchemically-synthesized oligonucleotides (see above) may be expressed inappropriate prokaryotic hosts. See Oppermann et al., U.S. Pat. No.5,324,819, which is herein incorporated by reference, for an exemplaryplasmid and protocol. An expression vector based on pBR322 andcontaining a synthetic trp promoter, operator and the modified trp LEleader can be opened at the EcoRI and PstI restriction sites, and aFB-FB COP gene fragment can be inserted between these sites, where FB isa fragment B of Staphylococcal Protein A. The expressed fusion proteinresults from attachment of the COP gene to a fragment encoding FB. TheCOP protein is joined to the leader protein via a hinge region havingthe sequence asp-pro-asn-gly. This hinge permits chemical cleavage ofthe fusion protein with dilute acid at the asp-pro site or cleavage atasn-gly with hydroxylamine. Cleavage at the hinge releases COP protein.

2. Mammalian Cell Expression

Recombinant production of mammalian proteins for therapeutic uses may beexpressed in mammalian cell culture systems in order to produce aprotein whose structure is most like that of the natural material.Recombinant protein production in mammalian cells requires theestablishment of appropriate cells and cell lines that are easy totransfect, are capable of stably maintaining foreign DNA with anunrearranged sequence, and which have the necessary cellular componentsfor efficient transcription, translation, post-translation modification,and secretion of the protein. In addition, a suitable vector carryingthe gene of interest is necessary.

DNA vector design for transfection into mammalian cells should includeappropriate sequences to promote expression of the gene of interest,including appropriate transcription initiation, termination, andenhancer sequences, as well as sequences that enhance translationefficiency, such as the Kozak consensus sequence. Preferred DNA vectorsalso include a marker gene and means for amplifying the copy number ofthe gene of interest.

Substantial progress in the development of mammalian cell expressionsystems has been made in the last decade and many aspects of the systemare well characterized. A detailed review of the state of the art of theproduction of foreign proteins in mammalian cells, including usefulcells, protein expression-promoting sequences, marker genes, and geneamplification methods, is disclosed in Bendig, Mary M., GeneticEngineering, 7, pp. 91-127 (1988).

Briefly, among the best characterized transcription promoters useful forexpressing a foreign gene in a particular mammalian cell are the SV40early promoter, the adenovirus promoter (AdMLP), the mousemetallothionein-I promoter (mMT-I), the Rous sarcoma virus (RSV) longterminal repeat (LTR), the mouse mammary tumor virus long terminalrepeat (MMTV-LTR), and the human cytomegalovirus majorintermediate-early promoter (hCMV). The DNA sequences for all of thesepromoters are known in the art and are available commercially.

One of the better characterized methods of gene amplification inmammalian cell systems is the use of the selectable dihydrofolatereductase (DHFR) gene in a dhfr-cell line. Generally, the DHFR gene isprovided on the vector carrying the gene of interest, and addition ofincreasing concentrations of the cytotoxic drug methotrexate leads toamplification of the DHFR gene copy number, as well as that of theassociated gene of interest. DHFR as a selectable, amplifiable markergene in transfected chinese hamster ovary cell lines (CHO cells) isparticularly well characterized in the art. Other useful amplifiablemarker genes include the adenosine deaminase (ADA) and glutaminesynthetase (GS) genes.

In the currently preferred expression system, gene amplification isfurther enhanced by modifying marker gene expression regulatorysequences (e.g., enhancer, promoter, and transcription or translationinitiation sequences) to reduce the levels of marker protein produced.Lowering the level of DHFR transcription has the effect of increasingthe DHFR gene copy number (and the associated OP-1 gene) in order for atransfected cell to adapt to grow in even low levels of methotrexate(MTX) (e.g., 0.1 μM MTX). Preferred expression vectors (pH754 andpH752), have been manipulated using standard recombinant DNA technology,to create a weak DHFR promoter. As will be appreciated by those skilledin the art, other useful weak promoters, different from those disclosedand preferred herein, can be constructed using standard vectorconstruction methodologies. In addition, other, different regulatorysequences also can be modified to achieve the same effect.

The choice of cells/cell lines is also important and depends on theneeds of the experimenter. Monkey kidney cells (COS) provide high levelsof transient gene expression, providing a useful means for rapidlytesting vector construction and the expression of cloned genes. COScells are transfected with a simian virus 40 (SV40) vector carrying thegene of interest. The transfected COS cells eventually die, thuspreventing the long term production of the desired protein product.However, transient expression does not require the time consumingprocess required for the development of a stable cell line.

Among established cell lines, CHO cells may be the best characterized todate, and are the currently preferred cell line for mammalian cellexpression of recombinant osteogenic protein. CHO cells are capable ofexpressing proteins from a broad range of cell types. The generalapplicability of CHO cells and its successful production for a widevariety of human proteins in unrelated cell types emphasizes theunderlying similarity of all mammalian cells. Thus, while theglycosylation pattern on a recombinant protein produced in a mammaliancell expression system may not be identical to the natural protein, thedifferences in oligosaccharide side chains are often not essential forbiological activity of the expressed protein.

The methodology disclosed herein includes the use of COS cells for therapid evaluation of vector construction and gene expression, and the useof established cell lines for long term protein production. Of the celllines disclosed, OP-1 expression from CHO cell lines currently is mostpreferred.

Several different mammalian cell expression systems have been used toexpress recombinant OP-1 proteins which may be used in concert with aMPSF according to this invention. In particular, COS cells are used forthe rapid assessment of vector construction and gene expression, usingan SV40 vector to transfect the DNA sequence into COS cells. Stable celllines are developed using CHO cells (chinese hamster ovary cells) and atemperature-sensitive strain of BSC cells (simian kidney cells,BSC40-tsA58; Biotechnology, 6, pp. 1192-96 (1988)) for the long termproduction of OP-1.

Two different promoters were found most useful to transcribe hOP1 (Seq.ID No. 1): the CMV promoter and the MMTV promoter, boosted by theenhancer sequence from the Rous sarcoma virus LTR. The mMT promoter(mouse metallothionein promoter) and the SV40 late promoter have alsobeen tested. Several selection marker genes also are used, namely, neo(neomycin) and DHFR.

The DHFR gene also may be used as part of a gene amplification schemefor CHO cells. Another gene amplification scheme relies on thetemperature sensitivity (ts) of BSC40-tsA58 cells transfected with anSV40 vector. Temperature reduction to 33° C. stabilizes the ts SV40 Tantigen which leads to the excision and amplification of the integratedtransfected vector DNA, thereby also amplifying the associated gene ofinterest.

Stable cell lines were established for CHO cells as well as BSC40-tsA58cells (hereinafter referred to as "BSC cells"). The various cells, celllines and DNA sequences chosen for mammalian cell expression of the OP-1proteins of this invention are well characterized in the art and arereadily available. Other promoters, selectable markers, geneamplification methods and cells also may be used to express the OP-1proteins of this invention, as well as other osteogenic proteins.Particular details of the transfection, expression, and purification ofrecombinant proteins are well documented in the art and are understoodby those having ordinary skill in the art. Further details on thevarious technical aspects of each of the steps used in recombinantproduction of foreign genes in mammalian cell expression systems can befound in a number of texts and laboratory manuals in the art. See, e.g.,F. M. Ausubel et al., ed., Current Protocols in Molecular Biology, JohnWiley & Sons, New York (1989).

a) Exemplary Expression Vectors

Restriction maps and sources of various exemplary expression vectorsdesigned for OP-1 expression in mammalian cells have been described(Oppermann et al., U.S. Pat. No. 5,354,557, incorporated herein byreference; see FIGS. 19 (A-F) and accompanying text). Each of thesevector constructs employs a full-length cDNA sequence ("hOP1"; Seq. IDNo. 1) originally isolated from a human cDNA library (placenta) andsubsequently cloned into a conventional pUC vector (pUC-18) using pUCpolylinker sequences at the insertion sites.

It will be appreciated by those skilled in the art that DNA sequencesencoding truncated forms of osteogenic protein may also be used in thesevectors, provided that the expression vector or host cell then providesthe sequences necessary to direct processing and secretion of theexpressed protein.

Each vector employs an SV40 origin of replication (ori), useful formediating plasmid replication in primate cells (e.g., COS and BSCcells). In addition, the early SV40 promoter is used to drivetranscription of marker genes on the vector (e.g., neo and DHFR).

The pH717 expression vector (FIG. 19A) contains the neomycin (neo) geneas a selection marker. This marker gene is well characterized in the artand is available commercially. Alternatively, other selectable markersmay be used. The particular vector used to provide the neo gene DNAfragment for pH717 may be obtained from Clontech, Inc., Palo Alto,Calif. (pMAM-neo-blue). This vector also may be used as the backbone. InpH717, hOP1 transcription is driven by the CMV promoter with RSV-LTR(Rous sarcoma virus long terminal repeat) and MMTV-LTR (mouse mammarytumor virus long terminal repeat) enhancer sequences. These sequencesare known in the art, and are available commercially. For example,vectors containing the CMV promoter sequence (e.g., pCDM8) may beobtained from Invitrogen Inc., San Diego, Calif.

Expression vector pH731 (FIG. 19B), utilizes the SV40 late promoter todrive hOP1 transcription. As indicated above, the sequence andcharacteristics of this promoter also are well known in the art. Forexample, pH731 may be generated by inserting the SmaI-BamHI fragment ofhOP1 into pEUK-C1 (Clontech, Inc., Palo Alto, Calif.).

The pH752 and pH754 expression vectors contain the DHFR gene under SV40early promoter control, as both a selection marker and as an induciblegene amplifier. The DNA sequence for DHFR is well characterized in theart, and is available commercially. For example, pH754 may be generatedfrom pMAM-neo (Clontech, Inc., Palo Alto, Calif.) by replacing the neogene (BamHI digest) with an SphI-BamHI, or a PvuII-BamHI fragment frompSV5-DHFR (ATCC #37148), which contains the DHFR gene under SV40 earlypromoter control. A BamHI site can be engineered at the SphI or PvuIIsite using standard techniques (e.g., by linker insertion orsite-directed mutagenesis) to allow insertion of the fragment into thevector backbone. hOP1 DNA can be inserted into the polylinker sitedownstream from the MMTV-LTR sequence, yielding pH752 (FIG. 19D). TheCMV promoter sequence then may be inserted into pH752 (e.g., from pCDM8,Invitrogen, Inc.), yielding pH754 (FIG. 19C).

The SV40 early promoter, which drives DHFR expression, is modified inthese vectors to reduce the level of DHFR mRNA produced. Specifically,the enhancer sequences and part of the promoter sequence have beendeleted, leaving only about 200 bases of the promoter sequence upstreamof the DHFR gene. Host cells transfected with these vectors are adaptedto grow in 0.1 μM MTX and can increase OP-1 production significantly(see, e.g., Table 8, Oppermann et al., U.S. Pat. No. 5,354,557).

The pW24 vector (FIG. 19E), is essentially identical in sequence top754, except that neo is used as the marker gene (see pH717)in place ofDHFR. Similarly, pH783 (FIG. 19F) contains the amplifiable marker DHFR,but here OP-1 is under mMT (mouse metallothionein promoter) control. ThemMT promoter is well characterized in the art and is availablecommercially.

All vectors tested are stable in the various cells used to express OP-1,and provide a range of OP-1 expression levels.

b) Exemplary Mammalian Cells

Recombinant OP-1 has been expressed in three different cell expressionsystems: COS cells for rapidly screening the functionality of thevarious expression vector constructs, CHO cells for the establishment ofstable cell lines, and BSC40-tsA58 cells as an alternative means ofproducing OP-1 protein. The CHO cell expression system disclosed hereinis contemplated to be the best mode currently known for long-termrecombinant OP-1 production in mammalian cells.

(1) COS Cells

COS cells (simian kidney cells) are used for rapid screening of vectorconstructs and for immediate, small scale production of OP-1 protein.COS cells are well known in the art and are available commercially. Theparticular cell line described herein may be obtained through theAmerican Type Culture Collection (ATCC #COS-1, CRL-1650).

OP-1 expression levels from these different expression vectors, analyzedby Northern and Western blot assays, are compared Oppermann et al. (seeTable 7, Oppermann et al.).

Large scale preparations of OP-1 from transfected COS cells may beproduced using conventional roller bottle technology. Briefly, 14×10⁶cells are used to seed each bottle. After 24 hrs of growth, the cellsare transfected with 10 μg of vector DNA (e.g., pH717) per 10⁶ cells,using the DEAE-dextran method. Cells are then conditioned in serum-freemedia for 120 hr before harvesting the media for protein analysis.Following this protocol, OP-1 yield is approximately 2-6 ng/ml.

(2) BSC CELLS

The BSC40-tsA58 cell line ("BSC cells") is a temperature-sensitive (ts)strain of simian kidney cells (Biotechnology, 6, pp. 1192-96 (1988))which overcomes some of the problems associated with COS cells. TheseBSC cells have the advantage of being able to amplify gene sequencesrapidly on a large scale with temperature downshift, without requiringthe addition of exogenous, potentially toxic drugs. In addition, afterinduction and stimulation of OP-1 expression, the cells may betransferred to new growth medium, grown to confluence at 39.5° C. andinduced a second time by downshifting the temperature to 33° C. BSCcells may be used to establish stable cell lines rapidly for proteinproduction.

OP-1 expression in transfected BSC cells may be induced by shifting thetemperature down to 33° C. in media containing 10% FCS, and harvestingthe conditioned media after 96 hrs of incubation. Comparable amounts ofOP-1 mRNA and protein are obtained, as compared with CHO cells (e.g.,100-150 ng OP-1/ml conditioned media from BSC clones transfected withpH717, see Oppermann et al.).

(3) CHO Cells

CHO cells (chinese hamster ovary cells) may be used for long term OP-1production and are the currently preferred cell line for mammalian cellexpression of OP-1. CHO cell lines are well characterized for the smalland large scale production of foreign genes and are availablecommercially. See Oppermann et al., U.S. Pat. No. 5,354,557,incorporated herein by reference, for a detailed description of:establishing a stable transfected cell line with high hOP-1 expressionlevels, subcloning transfected cells to obtain high expressionsubclones, characterizing subclone DNA insert copy numbers, andscreening subclones for OP-1 mRNA and protein expression levels.Oppermann et al. also provides a detailed description of a rapidpurification method for obtaining recombinantly produced OP-1 of about90% purity, and further data demonstrating the physical characteristics(molecular weight and glycosylation profiles) and osteogenic activitiesof a variety of recombinant forms of OP-1 expressed in the cell linesdescribed above.

Accordingly, it is anticipated that active mature OP-1 sequences,including full-length, truncated and mutationally-altered active formsof the protein, can be expressed from other different prokaryotic andeukaryotic cell expression systems using procedures essentially asdescribed herein. The proteins produced may have varying N-termini, andthose expressed from eukaryotic cells may have varying glycosylationpatterns. Finally, it will also be appreciated that these variations inthe recombinant osteogenic protein produced will be characteristic ofthe host cell expression system used rather than of the protein itself.

EXAMPLE 3

Synergistic Effect of Exogenous IGF-I on the OP-1-inducedDifferentiation of Fetal Rat Calvarial (FRC) Cells

Primary osteoblast cell cultures were prepared from fetal rat calvariausing published procedures (M. A. Aronow et al., J. Cell Physiol., 143,pp. 213-221 (1990); T. K. McCarthy et al., J. Bone Miner. Res., 3, pp.401-8 (1988)). Briefly, cells were harvested by sequential collagenasedigestions of the calvarium and cells from digestions III to V werepooled. Fetal rat calvaria (FRC) cells were plated in complete medium(MEM, alpha; GIBCO/BRL, Grand Island, N.Y.) containing 10% fetal bovineserum, vitamin C (100 μg/ml), and antibiotics (100 U/ml penicillin, and100 mg/ml streptomycin). Cultures were incubated at 37° C. with 95%air/5% CO₂ for several days to reach confluence. Cells were thensubcultured for experimentations.

FRC cells were subcultured in 48-well plates (COSTAR, Cambridge, Mass.)in complete MEM medium with 10% fetal bovine serum until confluent inabout 4 days. Confluent cells were rinsed with Hank's balanced saltsolution (HBSS) and treated with serum-free α-MEM medium (with 0.1% BSA,100 U/ml penicillin, and 100 mg/ml streptomycin) containing theappropriate solvent vehicle (50% acetonitrile/0.1% trifluoroacetic acidfor OP-1 treatment or 0.1N acetic acid for IGFI treatment) orrecombinant human OP-1, or IGFI at the concentrations indicated. Solventvehicle concentration in the culture medium never exceeded 0.1%. At theend of treatment, cells were lysed and total cellular alkalinephosphatase activity was measured (typically after 48 hours oftreatment).

Confluent FRC cells (6-8×10⁶ cells/T-150 flask) were rinsed once withHBSS to remove the complete medium and then incubated in serum-freeα-MEM medium (with 0.1% BSA, 100 U/ml penicillin, and 100 mg/mlstreptomycin) in the presence or absence of OP-1 for varying intervals.OP-1 was dissolved in 50% acetonitrile and 0.1% trifluoroacetic acid(TFA). At the end of treatments, cells in the T-150 flask were rinsedwith ice-cold PBS solution to remove serum-free medium and used forsubsequent RNA isolation.

Alkaline phosphatase Activity Assay

Total cellular alkaline phosphatase activity was determined using acommercial assay kit (Sigma, St. Louis, Mo.). Cell lysates were preparedby aspirating the medium from the 48-well plate, rinsing the cells withice-cold PBS, and lysing the cells with 0.05% Triton X-100 andsonication for 60 sec. Alkaline phosphatase activity in the lysates wasmeasured in 2-amino-2-methyl-1-propanol buffer (pH 10.3) withp-nitrophenyl phosphate as substrate at 37° C. Reactions were performedin 96-well plates for 1-2 h. Following color development, reactions wereterminated with 0.5N NaOH. Absorbance of the reaction was measured at405 nm using a Hewlett Packard Genenchem automatic plate reader. Totalprotein level in the lysates was measured according to Bradford (M.Bradford, Anal. Biochem., 72, pp.248-54 (1976)) using bovine serumalbumin as a standard. Alkaline phosphatase activity was expressed asnmol p-nitrophenol liberated per microgram of total cellular protein.

RNA isolation

Total RNA was isolated with cold Utraspec (Biotecx Lab., Houston, Tex.)following the manufacturer's recommendation. RNA was recovered byprecipitation and dissolved in DEPC-H₂ O. The amount of RNA recoveredwas estimated by A₂₆₀ reading. The integrity of the RNA preparation wasexamined by gel electrophoresis on 1% agarose. RNA was detected by EtBrstaining. Only RNA preparations showing intact species were used forsubsequent analyses.

Northern blot analysis

Total RNAs (20 μg) were denatured with formaldehyde and formamide at 65°C. for 15 min and analyzed on a 1% GTG agarose gel containing 2.2 Mformaldehyde. RNA standards (0.24-9.5 kb) from GIBCO/BRL (Grand Island,N.Y.) were used as size markers. The fractionated RNA was transferredonto `Nytran plus" membrane using a Turboblot apparatus (Schleicher &Schuell, Inc., Keene, N.H.). The lane containing the standards was cutfrom the blot and stained with methylene blue. The RNA was covalentlylinked to the membrane using a UV Crosslinker (Stratagene, La Jolla,Calif.). The membranes were hybridized overnight at 42° C. with theosteocalcin or type I collagen DNA probes, washed twice in 2×SSC at roomtemperature for 20 min each, twice in 2×SSC/1% SDS at 60° C. for 1 houreach, and finally twice in 0.1×SSC at room temperature for 30 min each.The blots were exposed to a PhosphorImage screen and analyzed asdescribed above. Four blots with different RNA preparations wererepeated for each probe.

Statistical Analysis

Multiple means were compared with one-way analysis of variance, followedby the student t-test for paired comparisons with the control, using theANOVA and T-Test programs in PSIPlot (Poly Software International, SaltLake City, Utah) for personal computers.

EXAMPLE 4 Identifying a First MPSF that Stimulates Tissue Induction by aMorphogenic Protein

The FRC cell alkaline phosphatase (AP) assay was performed as describedin Example 3 to test increasing concentrations of putative MPSFs incombination with a single concentration (200 ng/ml) of osteogenicprotein OP-1.

At least four experimental groups were tested: control cells treatedwith no OP-1 or MPSF; group I cells treated with increasingconcentrations of MPSF alone; group II cells treated with 200 ng/ml ofOP-1 alone; and group III cells, treated with 200 ng/ml OP-1 in thepresence of increasing concentrations of the MPSF.

FIG. 5 shows the effects of estradiol (0.05-5.0 nM; purchased fromSigma, St. Louis, Mo.) and 200 ng/ml of OP-1 on FRC cell alkalinephosphatase activity at 48 hours post-treatment. Estradiol alone did notappear to stimulate AP activity. In the presence of 0.5 nM estradiol and200 ng/ml of OP-1, the level of AP activity was almost eleven-foldhigher than the control, and about three-fold higher than cells treatedwith OP-1 alone.

FIG. 6 shows the effects of growth hormone (hGH; 10-1000 ng/ml;purchased from Sigma, St. Louis, Mo.) and 200 ng/ml of OP-1 on FRC cellalkaline phosphatase activity after 48 hours. All concentrations of hGHtested in the presence of 200 ng/ml of OP-1 stimulated the induction ofAP activity over that observed for OP-1 alone ("0"). Higher hGHconcentrations appeared to have more of a stimulatory effect than lowerconcentrations.

FIG. 7 shows the effects of hydrocortisone (HC; 0.05-5 nM; purchasedfrom Sigma, St. Louis, Mo.) and 200 ng/ml of OP-1 on FRC cell alkalinephosphatase activity after 48 hours. HC alone did not stimulate APactivity in FRC cells. In the presence of 0.5 nM HC and 200 ng/ml OP-1,the level of AP activity is about three-fold higher than in controlcells, and about two-fold higher than in cells treated with OP-1 alone.

FIG. 8 shows the effects of insulin (0.05-5 nM; purchased from Sigma,St. Louis, Mo.) and 200 ng/ml of OP-1 on FRC cell alkaline phosphataseactivity after 48 hours. Insulin alone did not stimulate AP activity inFRC cells. In the presence of 0.05 nM or 0.5 nM insulin and 200 ng/mlOP-1, the level of AP activity is about four-fold higher than in controlcells, and about two-fold higher than in cells treated with OP-1 alone.

FIG. 9 shows the effects of parathyroid hormone (PTH; 25-200 nM;purchased from Sigma, St. Louis, Mo.) and 200 ng/ml of OP-1 on FRC cellalkaline phosphatase activity after 48 hours. PTH alone did notstimulate AP activity in FRC cells. Low concentrations of PTH (25 and100 nM) and 200 ng/ml OP-1 appear to have no effect on OP-1-inducedstimulation of AP activity. In the presence of 200 nM PTH and 200 ng/mlOP-1, the level of AP activity is about five-fold higher than in controlcells, and about two-fold higher than in cells treated with OP-1 alone.

Finally, FIG. 10 shows the effects of progesterone (PG; 0.05-5 nM;purchased from Sigma, St. Louis, Mo.) and 200 ng/ml of OP-1 on FRC cellalkaline phosphatase activity after 48 hours. PG alone (5 nM) appears tostimulate AP activity about three-fold beyond control cells. PG (5 nM)in the presence of 200 ng/ml OP-1 appear to increase the level of APactivity about four-fold higher than in control cells, and abouttwo-fold higher than in cells treated with OP-1 alone.

EXAMPLE 5 Identifying Additional MPSFs that Stimulate Tissue Inductionby a Morphogenic Protein/MPSF Combination

Once an effective morphogenic protein/MPSF combination has beenidentified, one or more additional MPSFs that increase further thestimulation of tissue induction by that morphogenic protein/MPSFcombination may be identified. An assay done essentially according tothe procedures set forth in Examples 3 and 4 was performed except thatFRC cells were incubated with a combination of 200 ng/ml of OP-1 and 25ng/ml of IGF-1 in the presence or absence of increasing concentrationsof PTH (25-200 nM). The presence of PTH (at concentrations of at leastabout 50 nM) significantly increased the AP activity induced by theOP-1/IGF-I combination.

EXAMPLE 6 Preparation of Bone-Derived Matrices for Use in MorphogenicDevices

Demineralized bone matrix, preferably bovine bone matrix, is preparedusing previously published procedures (Sampath and Reddi, Proc. Natl.Acad. Sci. USA, 80, pp. 6591-95 (1983)), as described in Example 1.

Demineralized bone matrix is extracted with 5 volumes of 4Mguanidine-HCl, 50 mM Tris-HCl, pH 7.0 for 16 hr. at 4° C. The suspensionis filtered. The insoluble material is collected and used to fabricatethe matrix. The material is mostly collagenous in nature and is devoidof osteogenic or chondrogenic activity.

The major component of all bone matrices is Type-I collagen. In additionto collagen, demineralized bone extracted includes non-collagenousproteins which may account for 5% of its mass. In a xenogenic matrix,these non-collagenous components may present themselves as potentantigens, and may constitute immunogenic and/or inhibitory components.These components also may inhibit osteogenesis in allogenic implants byinterfering with the developmental cascade of bone differentiation.

Treatment of the matrix particles with a collagen fibril-modifying agentextracts potentially unwanted components from the matrix, and alters thesurface structure of the matrix material. Useful agents include acids,organic solvents or heated aqueous media. Various treatments aredescribed below. A detailed physical analysis of the effect thesefibril-modifying agents have on demineralized, quanidine-extracted bonecollagen particles is disclosed in U.S. Pat. No. 5,171,574, thedisclosure of which is hereby incorporated by reference.

After contact with the fibril-modifying agent, the treated matrix iswashed to remove any extracted components, following a form of theprocedure set forth below:

1. Suspend in TBS (Tris-buffered saline) 1 g/200 ml and stir at 4° C.for 2 hrs; or in 6M urea, 50 mM Tris-HCl, 500 mM NaCl, pH 7.0 (UTBS) orwater and stir at room temperature (RT) for 30 minutes (sufficient timeto neutralize the pH);

2. Centrifuge and repeat wash step; and

3. Centrifuge; discard supernatant; wash residue with water; andlyophilize.

Acid Treatments

1. TRIFLUOROACETIC ACID

Trifluoroacetic acid is a strong non-oxidizing acid that is a knownswelling agent for proteins, and which modifies collagen fibrils.

Bovine bone residue prepared as described above is sieved, and particlesof the appropriate size are collected. These particles are extractedwith various percentages (1.0% to 100%) of trifluoroacetic acid andwater (v/v) at 0° C. or at room temperature for 1-2 hours with constantstirring. The treated matrix is filtered, lyophilized, or washed withwater/salt and then lyophilized.

2. HYDROGEN FLUORIDE

Like trifluoroacetic acid, hydrogen fluoride (HF) is a strong acid andswelling agent, and also is capable of altering intraparticle surfacestructure. Hydrogen fluoride is also a known deglycosylating agent. Assuch, HF may function to increase the osteogenic activity of thesematrices by removing the antigenic carbohydrate content of anyglycoproteins still associated with the matrix after guanidineextraction.

Bovine bone residue prepared as described above is sieved, and particlesof the appropriate size are collected. The sample is dried in vacuo overP205, transferred to the reaction vessel and exposed to anhydroushydrogen fluoride (10-20 ml/g of matrix) by distillation onto the sampleat -70° C. The vessel is allowed to warm to 0° C. and the reactionmixture is stirred at this temperature for two hours. After evaporationof the hydrogen fluoride in vacuo, the residue is dried thoroughly invacuo over KOH pellets to remove any remaining traces of acid. Extent ofdeglycosylation can be determined from carbohydrate analysis of matrixsamples taken before and after treatment with hydrogen fluoride, afterwashing the samples appropriately to remove non-covalently boundcarbohydrates. SDS-extracted protein from HF-treated material isnegative for carbohydrate as determined by Con A blotting.

The deglycosylated bone matrix is next washed twice in TBS(Tris-buffered saline) or UTBS, water-washed, and then lyophilized.

Other acid treatments are envisioned in addition to HF and TFA. TFA is acurrently preferred acidifying reagent in these treatments because ofits volatility. However, it is understood that other, potentially lesscaustic acids may be used, such as acetic or formic acid.

Solvent Treatments

1. DICHLOROMETHANE

Dichloromethane (DCM) is an organic solvent capable of denaturingproteins without affecting their primary structure. This swelling agentis a common reagent in automated peptide synthesis, and is used inwashing steps to remove components. Bovine bone residue, prepared asdescribed above, is sieved, and particles of the appropriate size areincubated in 100% DCM or, preferably, 99.9% DCM/0.1% TFA. The matrix isincubated with the swelling agent for one or two hours at 0° C. or atroom temperature. Alternatively, the matrix is treated with the agent atleast three times with short washes (20 minutes each) with noincubation.

2. ACETONITRILE

Acetonitrile (ACN) is an organic solvent capable of denaturing proteinswithout affecting their primary structure. It is a common reagent usedin high-performance liquid chromatography, and is used to elute proteinsfrom silica-based columns by perturbing hydrophobic interactions.

Bovine bone residue particles of the appropriate size, prepared asdescribed above, are treated with 100% ACN (1.0 g/30 ml) or, preferably,99.9% ACN/0.1% TFA at room temperature for 1-2 hours with constantstirring. The treated matrix is then water-washed, or washed with ureabuffer or 4M NaCl, and lyophilized. Alternatively, the ACN or ACN/TFAtreated matrix may be lyophilized without wash.

3. ISOPROPANOL

Isopropanol is also an organic solvent capable of denaturing proteinswithout affecting their primary structure. It is a common reagent usedto elute proteins from silica HPLC columns. Bovine bone residueparticles of the appropriate size prepared as described above aretreated with 100% isopropanol (1.0 g/30 ml) or, preferably, in thepresence of 0.1% TFA, at room temperature for 1-2 hours with constantstirring. The matrix is then water-washed or washed with urea buffer or4M NaCl before being lyophilized.

4. CHLOROFORM

Chloroform also may be used to increase surface area of bone matrix likethe reagents set forth above, either alone or acidified. Treatment asdescribed above is effective to assure that the material is free ofpathogens prior to implantation.

Heat Treatment

The currently most preferred agent is a heated aqueous fibril-modifyingmedium such as water, to increase the matrix particle surface area andporosity. The currently most preferred aqueous medium is an acidicaqueous medium having a pH of less than about 4.5, e.g., within therange of about pH 2-pH 4 which may help to "swell" the collagen beforeheating. Acetic acid (0.1%), which has a pH of about 3, currently ismost preferred. 0.1M acetic acid also may be used.

Various amounts of delipidated, demineralized guanidine-extracted bonecollagen are heated in the aqueous medium (1 g matrix/30 ml aqueousmedium) under constant stirring in a water jacketed glass flask, andmaintained at a given temperature for a predetermined period of time.Preferred treatment times are about one hour, although exposure times ofbetween about 0.5 to two hours appear acceptable. The temperatureemployed is held constant at a temperature within the range of about 37°C. to 65° C. The currently preferred heat treatment temperature iswithin the range of about 45° C. to 60° C.

After the heat treatment, the matrix is filtered, washed, lyophilizedand used for implantation. Where an acidic aqueous medium is used, thematrix also is preferably neutralized prior to washing andlyophilization. A currently preferred neutralization buffer is a 200 mMsodium phosphate buffer, pH 7.0. To neutralize the matrix, the matrixpreferably is first allowed to cool following thermal treatment, theacidic aqueous medium (e.g., 0.1% acetic acid) is then removed andreplaced with the neutralization buffer and the matrix agitated forabout 30 minutes. The neutralization buffer may then be removed and thematrix washed and lyophilized (see infra).

The effects of heat treatment on morphology of the matrix material isdescribed in Oppermann, et. al., U.S. Pat. No. 5,354,557. Hot aqueoustreatment can increase the degree of micropitting on the particlesurface (e.g., about 10-fold,) as well as also substantially increasingthe particle's porosity. This alteration of the matrix particle'smorphology substantially increases the particle surface area. Carefulmeasurement of the pore and micropit sizes reveals that hot aqueousmedium treatment of the matrix particles yields particle pore andmicropit diameters within the range of 1 μm to 100 μm.

Oppermann et al. also show that a complete solvent extract from hotwater-treated matrix inhibits OP-1 induced new bone formation in a dosedependent manner. Thus such treatment may also be removing component(s)whose association with the matrix may interfere with new bone formationin vivo.

The matrix also may be treated to remove contaminating heavy metals,such as by exposing the matrix to a metal ion chelator. For example,following thermal treatment with 0.1% acetic acid, the matrix may beneutralized in a neutralization buffer containing sodium EDTA, e.g., 200mM sodium phosphate, 5 mM EDTA, pH 7.0. The use of 5 mM EDTA providesabout a 100-fold molar excess of chelator to residual heavy metalspresent in the most contaminated matrix tested to date. Subsequentwashing of the matrix following neutralization appears to remove thebulk of the EDTA. EDTA treatment of matrix particles reduces theresidual heavy metal content of all metals tested (Sb, As, Be, Cd, Cr,Cu, Co, Pb, Hg, Ni, Se, Ag, Zn, Tl) to less than about 1 ppm. Bioassayswith EDTA-treated matrices indicate that treatment with the metal ionchelator does not inhibit bone inducing activity.

The collagen matrix materials preferably take the form of a fine powder,insoluble in water, comprising nonadherent particles. It may be usedsimply by packing into the volume where new bone growth or sustainedrelease is desired, held in place by surrounding tissue. Alternatively,the powder may be encapsulated in, e.g., a gelatin or polylactic acidcoating, which is absorbed readily by the body. The powder may be shapedto a volume of given dimensions and held in that shape by interadheringthe particles using, for example, soluble, species-biocompatiblecollagen. The material may also be produced in sheet, rod, bead, orother macroscopic shapes.

Demineralized rat bone matrix used as an allogenic matrix may beprepared from several of the dehydrated diaphyseal shafts of rat femurand tibia (as described in Oppermann et al., U.S. Pat. No. 5,354,557,which is incorporated herein by reference) to produce a bone particlesize that passes through a 420 μm sieve. The bone particles aresubjected to dissociative extraction with 4M guanidine-HCl. Suchtreatment results in a complete loss of the inherent ability of the bonematrix to induce endochondral bone differentiation. The remaininginsoluble material is used to fabricate the matrix. The material ismostly collagenous in nature, and upon implantation, does not inducecartilage and bone formation. All new preparations are tested formineral content and osteogenic activity before use. The total loss ofbiological activity of bone matrix is restored when an activemorphogenic protein fraction or a substantially pure morphogenic proteinpreparation is reconstituted with the biologically inactive insolublecollagenous matrix.

ETHANOL TRIFLUOROACETIC ACID LYOPHILIZATION

In this procedure, morphogenic protein is solubilized in anethanol-trifluoroacetic acid solution (47.5% EtOH/0.01% TFA) and addedto the carrier material with the MPSF. Samples are vortexed and thenlyophilized. This method is currently preferred.

ACETONITRILE TRIFLUOROACETIC ACID LYOPHILIZATION

This is a variation of the above procedure, using anacetonitrile-trifluoroacetic acid (ACN/TFA) solution to solubilize themorphogenic protein that is then added to the MPSF and the carriermaterial. Samples are vigorously vortexed many times and thenlyophilized.

ETHANOL PRECIPITATION

Matrix is added to morphogenic protein and MPSF dissolved inguanidine-HCl. Samples are vortexed and incubated at a low temperature(e.g., 4° C.). Samples are then further vortexed. Cold absolute ethanol(5 volumes) is added to the mixture which is then stirred and incubated,preferably for 30 minutes at -20° C. After centrifugation (microfuge,high speed), the supernatant is discarded. The reconstituted matrix iswashed twice with cold concentrated ethanol in water (85% EtOH) and thenlyophilized.

UREA LYOPHILIZATION

For those morphogenic proteins that are prepared in urea buffer, theprotein is mixed with the MPSF and the matrix material, gently vortexedand then lyophilized. The lyophilized material may be used "as is" forimplants.

BUFFERED SALINE LYOPHILIZATION

Morphogenic protein preparations in physiological saline may also bevortexed with the MPSF and the matrix and lyophilized to producemorphogenically active material.

These procedures also can be used to adsorb other active therapeuticdrugs, hormones, and various bioactive species to the matrix forsustained release purposes.

EXAMPLE 7 Rat Model Bioassay for Bone Induction

This assay consists of implanting allogenic or xenogenic test samples insubcutaneous sites in recipient rats under ether anesthesia. MaleLong-Evans rats, aged 28-32 days, may be used. A vertical incision (1cm) is made under sterile conditions in the skin over the thoracicregion, and a pocket is prepared by blunt dissection. Approximately 25mg of the test sample is implanted deep into the pocket and the incisionis closed with a metallic skin clip. The day of implantation isdesignated as day one of the experiment. Implants are removed on day 12.The heterotropic site allows for the study of bone induction without thepossible ambiguities resulting from the use of orthotropic sites.

Bone inducing activity is determined biochemically by the specificactivity of alkaline phosphatase and calcium content of the day 12implant. An increase in the specific activity of alkaline phosphataseindicates the onset of bone formation. Calcium content, on the otherhand, is proportional to the amount of bone formed in the implant. Boneformation therefore is calculated by determining the calcium content ofthe implant on day 12 in rats and is expressed as "bone forming units,"where one bone forming unit represents the amount of protein that isneeded for half maximal bone forming activity of the implant on day 12.Bone induction exhibited by intact demineralized rat bone matrix isconsidered to be the maximal bone differentiation activity forcomparison purposes in this assay.

Cellular events during endochondral bone formation

Successful implants exhibit a controlled progression through the stagesof protein-induced endochondral bone development, including: (1)transient infiltration by polymorphonuclear leukocytes on day one; (2)mesenchymal cell migration and proliferation on days two and three; (3)chondrocyte appearance on days five and six; (4) cartilage matrixformation on day seven; (5) cartilage calcification on day eight; (6)vascular invasion, appearance of osteoblasts, and formation of new boneon days nine and ten; (7) appearance of osteoclasts, bone remodeling anddissolution of the implanted matrix on days twelve to eighteen; and (8)hematopoietic bone marrow differentiation in the ossicles on daytwenty-one. This time course in rats may be accelerated by increasingthe amounts of OP-1 added. It is possible that increasing amounts of oneor more MPSFs may also accelerate this time course. The shape of the newbone conforms to the shape of the implanted matrix.

Histological evaluation

Histological sectioning and staining is preferred to determine theextent of osteogenesis in the implants. Implants are fixed in BouinsSolution, embedded in paraffin, and cut into 6-8 μm sections. Stainingwith toluidine blue or hemotoxylin/eosin demonstrates clearly theultimate development of endochondral bone. Twelve-day implants areusually sufficient to determine whether the implants containnewly-induced bone.

Biological markers

Alkaline phosphatase (AP)activity may be used as a marker forosteogenesis. The enzyme activity may be determinedspectrophotometrically after homogenization of the implant. The activitypeaks at 9-10 days in vivo and thereafter slowly declines. Implantsshowing no bone development by histology have little or no alkalinephosphatase activity under these assay conditions. The assay is usefulfor quantification and obtaining an estimate of bone formation quicklyafter the implants are removed from the rat. Alternatively, the amountof bone formation can be determined by measuring the calcium content ofthe implant.

Gene expression patterns that correlate with endochondral bone or othertypes of tissue formation can also be monitored by quantitating mRNAlevels using procedures known to those of skill in the art such asNorthern Blot analysis. Such developmental gene expression markers maybe used to determine progression through tissue differentiation pathwaysafter osteogenic protein/MPSF treatments. These markers includeosteoblastic-related matrix proteins such as procollagen α₂ (I),procollagen α₁ (I), procollagen α₁ (III), osteonectin, osteopontin,biglycan, and alkaline phosphatase for bone regeneration (see e.g., Suvaet al., J. Bone Miner. Res., 8, pp. 379-88 (1993); Benayahu et al., J.Cell. Biochem., 56, pp. 62-73 (1994)).

EXAMPLE 8 Feline Model Bioassay for Bone Repair

A femoral osteotomy defect is surgically prepared. Without furtherintervention, the simulated fracture defect would consistently progressto non-union. The effects of osteogenic compositions and devicesimplanted into the created bone defects are evaluated by the followingstudy protocol.

The 1 cm and 2 cm femoral defect cat studies demonstrate that devicescomprising a matrix containing disposed osteogenic protein and MPSF can:(1) repair a weight-bearing bone defect in a large animal; (2)consistently induce bone formation shortly following (less than twoweeks) implantation; and (3) induce bone by endochondral ossification,with a strength equal to normal bone, on a volume for volume basis.Furthermore, all animals remain healthy during the study and show noevidence of clinical or histological laboratory reaction to theimplanted device. In this bone defect model, there is little or nohealing at control bone implant sites. The results provide evidence forthe successful use of the osteogenic compositions and devices of thisinvention to repair large, non-union bone defects.

Briefly, the procedure is as follows: Sixteen adult cats each weighingless than 10 lbs. undergo unilateral preparation of a 1 cm bone defectin the right femur through a lateral surgical approach. In otherexperiments, a 2 cm bone defect may be created. The femur is immediatelyinternally fixed by lateral placement of an 8-hole plate to preserve theexact dimensions of the defect.

Three different types of materials may be implanted in the surgicallycreated cat femoral defects: group I is a negative control group whichundergoes the same plate fixation with implants of 4Mguanidine-HCl-treated (inactivated) cat demineralized bone matrix powder(GuHCl-DBM) (360 mg); group II is a positive control group implantedwith biologically active demineralized bone matrix powder (DBM) (360mg); and groups III and IV undergo a procedure identical to groups I-II,with the addition of morphogenic protein alone (group III) andmorphogenic protein+MPSF (group IV) onto each of the GuHCl-DBM carriersamples.

All animals are allowed to ambulate ad libitum within their cagespost-operatively. All cats are injected with tetracycline (25 mg/kgsubcutaneously (SQ) each week for four weeks) for bone labeling. All butfour group III and four group IV animals are sacrificed four monthsafter femoral osteotomy.

In vivo radiomorphometric studies are carried out immediately post-op at4, 8, 12 and 16 weeks by taking a standardized X-ray of thelightly-anesthetized animal positioned in a cushioned X-ray jig designedto consistently produce a true anterio-posterior view of the femur andthe osteotomy site. All X-rays are taken in exactly the same fashion andin exactly the same position on each animal. Bone repair is calculatedas a function of mineralization by means of random point analysis. Afinal specimen radiographic study of the excised bone is taken in twoplanes after sacrifice.

At 16 weeks, the percentage of groups III and IV femurs that are united,and the average percent bone defect regeneration in groups I-IV arecompared. The group I GuHCl-DMB negative-control implants shouldgenerally exhibit no bone growth at four weeks, less than 10% at eightand 12 weeks, and about 16% (+/-10%) at 16 weeks. The group II DMBpositive-control implants should generally exhibit about 15-20% repairat four weeks, 35% at eight weeks, 50% (+/-10%) at 12 weeks and 70%(+/-12%) by 16 weeks.

Excised test and normal femurs may be immediately studied by bonedensitometry, or wrapped in two layers of saline-soaked towels, placedinto sealed plastic bags, and stored at -20° C. until further study.Bone repair strength, load-to-failure, and work-to-failure are tested byloading to failure on a specially designed steel 4-point bending jigattached to an Instron testing machine to quantitate bone strength,stiffness, energy absorbed and deformation to failure. The study of testfemurs and normal femurs yields the bone strength (load) in pounds andwork-to-failure in joules. Normal femurs exhibit a strength of 96(+/-12) pounds. Osteogenic device-implanted femur strength should becorrected for surface area at the site of fracture (due to the"hourglass" shape of the bone defect repair). With this correction, theresult should correlate closely with normal bone strength.

Following biomechanical testing, the bones are immediately sliced intotwo longitudinal sections at the defect site, weighed, and the volumemeasured. One-half is fixed for standard calcified bonehistomorphometrics with fluorescent stain incorporation evaluation, andone-half is fixed for decalcified hemotoxylin/eosin stain histologypreparation.

Selected specimens from the bone repair site are homogenized in cold0.15 M NaCl, 3 mM NaHCO₃, pH 9.0 by a Spex freezer mill. The alkalinephosphatase activity of the supernatant and total calcium content of theacid soluble fraction of sediment are then determined.

EXAMPLE 9 Rabbit Model Bioassay for Bone Repair

This assay is described in detail in Oppermann et al., U.S. Pat. No.5,354,557; see also Cook et al., J. of Bone and Joint Surgery, 76-A, pp.827-38 (1994), which are incorporated herein by reference). Ulnarnon-union defects of 1.5 cm are created in mature (less than 10 lbs) NewZealand White rabbits with epiphyseal closure documented by X-ray. Theexperiment may include implantation of devices into at least eightrabbits per group as follows: group I negative control implants of 4Mguanidine-HCl-treated (inactivated) demineralized bone matrix powder(GuHCl-DBM); group II positive control implants with biologically activedemineralized bone matrix powder (DBM); group III implants withosteogenic protein alone; group IV implants with osteogenic protein/MPSFcombinations, and group V controls receiving no implant. Ulnae defectsare followed for the full course of the eight week study in each groupof rabbits.

In another experiment, the marrow cavity of the 1.5 cm ulnar defect ispacked with activated osteogenic protein in rabbit bone powder in thepresence or absence of a MPSF. The bones are allografted in anintercalary fashion. Negative control ulnae are not healed by eightweeks and reveal the classic "ivory" appearance. In distinct contrast,the osteogenic protein/MPSF-treated implants "disappear"radiographically by four weeks with the start of remineralization by sixto eight weeks. These allografts heal at each end with mildproliferative bone formation by eight weeks. This type of device servesto accelerate allograft repair.

Implants treated with osteogenic protein in the presence of a MPSF mayshow accelerated repair, or may function at the same rate using lowerconcentrations of the osteogenic protein. As was described above, therabbit model may also be used to test the efficacy of and to optimizeconditions under which a particular osteogenic protein/MPSF combinationcan induce local bone and cartilage formation.

EXAMPLE 10 Dog Ulnar Defect Bioassay for Bone Repair

This assay is performed essentially as described in Cook et al.,Clinical Orthopaedics and Related Research, 301, pp. 302-112 (1994),which is incorporated herein by reference). Briefly, an ulnar segmentaldefect model is used to evaluate bone healing in 35-45 kg adult maledogs. Experimental composites comprising 500 mg of insoluble bovine bonecollagen are reconstituted with either 0, 625, 1200 or 2500 μg of OP-1(preferably recombinant OP-1 expressed in CHO cells; Example 2B) in theabsence or presence of increasing concentrations of one or more putativeMPSFs. Any osteogenic protein may be used in place of OP-1 in thisassay. Implantations at defect sites are performed with one carriercontrol and with the experimental series of OP-1 and OP-1/MPSFcombinations being tested. mechanical testing is performed on ulnae ofanimals receiving composites at 12 weeks after implantation. Radiographsof the forelimbs are obtained weekly until the animals are sacrificed ateither 12 or 16 postoperative weeks. Histological sections are analyzedfrom the defect site and from adjacent normal bone.

The presence of one or more MPSFs can increase the rate of bone repairin dog. The presence of one or more MPSFs may also permit the use ofreduced concentrations of osteogenic protein per composite to achievesimilar or the same results.

EXAMPLE 11 Monkey Ulnar and Tibial Defect Bioassay for Bone Repair

This bone healing assay in African green monkeys is performedessentially as described in Cook et al., J. Bone and Joint Surgery, 77A,pp. 734-50 (1995), which is incorporated herein by reference. Briefly, a2.0 cm osteoperiosteal defect is created in the middle of the ulnarshaft and filled with an implant comprising various matrices containing1000 μg of OP-1 (preferably recombinant OP-1 expressed in CHO cells;Example 2B) in the absence or presence of increasing concentrations ofone or more putative MPSFs. Experimental composites comprising variousmatrices reconstituted with either 0, 250, 500 or 100 or 2000 μg of OP-1in the absence or presence of increasing concentrations of one or moreputative MPSFs were used to fill 2.0 cm osteoperiosteal defects createdin the diaphysis of the tibia. Any osteogenic protein may be used inplace of OP-1 in this assay. Implantations at defect sites are performedwith one carrier control and with the experimental series of OP-1 andOP-1/MPSF combinations being tested. mechanical testing is performed onulnae and tibia of animals receiving composites. Radiographs andhistological sections are analyzed from the defect sites and fromadjacent normal bone as described in Cook et al.

The presence of one or more MPSFs can increase the rate of bone repairin the monkey. The presence of one or more MPSFs may also permit the useof reduced concentrations of osteogenic protein per composite to achievesimilar or the same results.

EXAMPLE 12 Rat Model Bioassay for Tendon/ligament-like Tissue Formation

The Sampath Reddi rat ectopic implant assay is modified such that theethanol precipitation step is substituted with a dialysis step againstwater if the morphogenic protein/MPSF composition is a solution, or adiafiltering step against water if it is a suspension, followed byequilibration to 0.1% trifluoroacetic acid. The resulting solution ismixed with 20 mg of rat matrix, the mixture frozen, lyophilized andenclosed in #5 gelatin capsules (or other functionally equivalentdevices). These devices are then implanted subcutaneously into abdominalthoracic region of rats (21-49 day old Male Long Evans rats wereemployed in Celeste et al.).

Subcutaneous implants are removed after ten days, and a section of eachis processed using known procedures for histological analysis (see e.g.,Ham and Cormack, Histology pp. 367-69 (J. B. Lippincott Co. 1979) (thedisclosure of which is hereby incorporated by reference)).Glycolmethacrylate sections (1 μm) are stained with Von Kossa and acidfuschin to visualize and quantitate the amount of embryonictendon/ligament-like tissue induced in each implant. Positive (e.g.,containing BMP-12) and negative (e.g., a mock device) implant controlgroups are compared to experimental implants comprising either amorphogenic protein alone, or a morphogenic protein in combination witha MPSF. Embryonic tendon/ligament-like tissue, characterized bytightly-packed fibroblast bundles oriented in the same plane, can beobserved in positive control implants after ten days.

EXAMPLE 13 Rat Model Bioassay for Nerve Regeneration and Repair

A matrix carrier is prepared. Wang et al. (WO 95/05846) used Collastat®,a collagen sponge (Vitaphore Wound Healing, Inc.), but any other desiredcarrier, such as those described herein, may be tested forapplicability. The collagen carrier is prepared by washing,lyophilizing, sterilizing and degassing, and is then loaded with, forexample, either: with no morphogenic protein (negative control group),with morphogenic protein only (group I), or with a particularcombination of morphogenic protein/MPSF (group II). Variations on theexperimental design allow one skilled in the art to test a variety ofdifferent morphogenic protein/MPSF combinations under variousconditions.

All manipulations are performed under sterile conditions. The loadedmatrices are placed inside approximately 1.6×20 mm lengths of sterilevented silastic or biodegradable tubing (stents) which may be trimmed toremove excess tubing before surgery. Vented silastic or biodegradablestents containing the matrices are applied microscopically andanastomized to the severed nerve endings, which are inserted into thestent for about 1 mm at each end, leaving a 15 mm "nerve defect" gap.Rats are tested for electrical return of function over a time course ofweeks after implantation. Compound muscle action potentials (CMAPs)provide a reproducible transcutaneous measurement for assessing thedegree of functional return. CMAP amplitude and latency is proportionalto the number of reinnervated axon/motor endplates and thus serves as auseful index of neuronal regeneration.

Animals may be sacrificed for histopathological examination at varioustimes post-implantation. Control stents implanted within subcutaneoustissues serve as histochemical controls.

What is claimed is:
 1. A method for improving the tissue inductiveactivity of a morphogenic protein on a mammalian progenitor cellcomprising the step of coadministering to the cell a morphogenic proteinstimulatory factor (MPSF) selected from the group consisting ofhormones, cytokines, peptides and growth factors, whereby the MPSFstimulates tissue induction by the morphogenic protein;with the provisothat when the progenitor cell is an osteoblast stimulated to form boneand the morphogenic protein is activin, the MPSF may not be estrogen orcalcitonin; when the progenitor cell is an osteoblast stimulated to formbone and the morphogenic protein is a BMP homodimer or TGF-β, the MPSFmay not be FGF, IGF-II, PDGF, estrogen, calcitonin or vitamin D; whenthe progenitor cell is an osteoblast stimulated to form bone orcartilage and the morphogenic protein is a BMP homodimer, the MPSF maynot be TGF-β; and when the progenitor cell is an osteoblast stimulatedto form bone and the morphogenic protein is a homodimer of BMP-2 orBMP-3, the MPSF may not be parathyroid hormone.
 2. The method accordingto claim 1, wherein the MPSF has an additive effect on stimulatingtissue induction by the morphogenic protein.
 3. The method according toclaim 1, wherein the MPSF has a synergistic effect on stimulating tissueinduction by the morphogenic protein.
 4. The method according to claim1, wherein the morphogenic protein is an osteogenic protein that iscapable of inducing the progenitor cell to form endochondral orintramembranous bone.
 5. The method according to claim 1, wherein themorphogenic protein is an osteogenic protein that is capable of inducingthe progenitor cell to form cartilage.
 6. The method according to claim1, wherein the morphogenic protein is an osteogenic protein that iscapable of inducing the progenitor cell to form tendon/ligament-like orneural tissue.
 7. The method according to claim 1, wherein themorphogenic protein comprises a polypeptide selected from the groupconsisting of: BMP-2, BMP-4, BMP-5, BMP-6, OP-1 (BMP-7), BMP-8, BMP-9,BMP-10, BMP-11, BMP-12, and BMP-13, COP-5 and COP-7.
 8. The methodaccording to claim 1, wherein the morphogenic protein comprises adisulfide bonded dimeric species comprising a polypeptide selected fromthe group consisting of OP-1, BMP-2, BMP-4 and BMP-6.
 9. The methodaccording to claim 1, wherein the morphogenic protein comprises OP-1.10. The method according to claim 1, wherein the morphogenic protein isproduced by the expression of a recombinant DNA molecule in a host cell.11. The method according to claim 1, wherein the morphogenic proteinstimulatory factor comprises at least one compound selected from thegroup consisting of: insulin-like growth factor I (IGF-I), estradiol,growth hormone (GH), growth and differentiation factor (GDF),hydrocortisone (HC), insulin, progesterone, parathyroid hormone (PTH),vitamin D, retinoic acid and IL-6.
 12. The method according to claim 1,wherein the MPSF is administered in an amount capable of synergisticallystimulating the ability of the morphogenic protein to induce tissueformation in the mammal.
 13. The method according to claim 1, whereinthe morphogenic protein stimulatory factor comprises an agent thatincreases IGF-I bioactivity in the mammal.
 14. The method according toclaim 1, wherein the morphogenic protein is present at a concentrationof at least about 1 ng/ml, and the morphogenic protein stimulatoryfactor is present at a concentration of at least about 0.01 ng/ml. 15.The method according to claim 1, wherein the morphogenic proteincomprises OP-1 at a concentration of from about 1 ng/ml to about 500ng/ml and the morphogenic protein stimulatory factor comprises IGF-I ata concentration from about 0.1 ng/ml to about 50 ng/ml.
 16. The methodaccording to claim 1, wherein the morphogenic protein comprises OP-1 ata concentration of from about 1 ng/ml to about 500 ng/ml and themorphogenic protein stimulatory factor comprises estradiol at aconcentration of from about 0.05 nM to about 1000 nM.
 17. The methodaccording to claim 1, wherein the morphogenic protein comprises OP-1 ata concentration of from about 1 ng/ml to about 500 ng/ml and themorphogenic protein stimulatory factor comprises growth hormone at aconcentration of from about 5 ng/ml to about 1000 ng/ml.
 18. The methodaccording to claim 1, wherein the morphogenic protein comprises OP-1 ata concentration of from about 1 ng/ml to about 500 ng/ml and themorphogenic protein stimulatory factor comprises hydrocortisone at aconcentration of from about 0.05 nM to about 5.0 nM.
 19. The methodaccording to claim 1, wherein the morphogenic protein comprises OP-1 ata concentration of from about 1 ng/ml to about 500 ng/ml and themorphogenic protein stimulatory factor comprises insulin at aconcentration of from about 0.01 nM to about 1000 nM.
 20. The methodaccording to claim 1, wherein the morphogenic protein comprises OP-1 ata concentration of from about 1 ng/ml to about 500 ng/ml and themorphogenic protein stimulatory factor comprises parathyroid hormone ata concentration of from about 10 nM to about 1000 nM.
 21. The methodaccording to claim 1, wherein the morphogenic protein comprises OP-1 ata concentration of from about 1 ng/ml to about 500 ng/ml and themorphogenic protein stimulatory factor comprises progesterone at aconcentration of from about 0.05 nM to about 1000 nM.